Hydroxyapatite-targeting multiarm polymers and conjugates made therefrom

ABSTRACT

The present invention provides, among other things, polymeric reagents suitable for reaction with biologically active agents to form conjugates, the polymeric reagents comprising one or more polymer chains and a plurality of hydroxyapatite-targeting moieties, and optionally the reagents include one or more degradable linkages that serve to divide the polymer chains into polymer segments having a molecular weight suitable for renal clearance.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/865,021, filed Apr. 17, 2013, now U.S. Pat. No. 8,815,227,which is a divisional application of U.S. patent application Ser. No.12/738,980, filed Jul. 28, 2010, now U.S. Pat. No. 8,440,787, which is a35 U.S.C. §371 application of International Application No.PCT/US2008/012091, filed Oct. 23, 2008, designating the United States,which claims the benefit of priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 60/982,012, filed Oct. 23, 2007, allof which are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

Among other things, this invention relates to water-soluble,non-peptidic polymers, and conjugates made therefrom, wherein thepolymer comprises at least one hydroxyapatite-targeting moiety.

BACKGROUND OF THE INVENTION

Covalent attachment of hydrophilic polymers to molecules havingpharmaceutically useful properties is of considerable utility for drugdelivery. There is a growing list of polymers whose conjugates haveentered clinical trials. Among them are conjugates of polyethyleneglycol, abbreviated as “PEG,” [Greenwald et al. (2003) Effective drugdelivery by PEGylated drug conjugates. Adv. Drug Delivery Rev.55:217-250; Harris et al. (2003) Effect of PEGylation onPharmaceuticals. Nat. Rev. Drug Discovery 2:214-221)],hydroxyethylcellulose, abbreviated as “HES,” (WO 2006/050959),poly(L-glutamic acid) [Li (2002) Poly(L-glutamic acid)-anticancer drugconjugates. Adv. Drug Delivery Rev. 54: 695-713]. PEG conjugates havebeen remarkably successful as several are marketed drugs (e.g. CIMZIA®,NEULASTA®, MACUGEN®, SOMAVERT®, PEGASYS®, and PEG-INTRON®). PEG is apolymer that possesses many beneficial properties. For instance, PEG issoluble in water and in many organic solvents, is non-toxic andnon-immunogenic, and when attached to a surface, PEG provides abiocompatible, protective coating. Common applications or uses of PEGinclude (i) covalent attachment to proteins to, for example, extendplasma half-life and reduce clearance through the kidney, (ii)attachment to surfaces such as in arterial replacements, bloodcontacting devices, and biosensors, (iii) use as a soluble carrier forbiopolymer synthesis, and (iv) use as a reagent in the preparation ofhydrogels. The other commonly used hydrophilic polymers claim similarproperties and potential uses.

In many if not all of the uses noted above, it is necessary to firstactivate the hydrophilic polymer by converting its active terminus,e.g., a hydroxyl group in the case of a PEG, to a functional groupcapable of readily reacting with a functional group found within adesired target molecule or surface, such as a functional group found onthe surface of a protein. For proteins, typical functional groupsinclude functional groups associated with the side chains of lysine,cysteine, histidine, arginine, aspartic acid, glutamic acid, serine,threonine, and tyrosine, as well as the N-terminal amino functionalgroup and the C-terminal carboxylic acid functional group. Othernontoxic biocompatible hydrophilic polymers may be substituted and aregenerally acceptable alternatives, with modest changes based on thespecific functional groups that are available for use in polymermodification and ultimately conjugation.

Using PEG as representative of the class, PEG used as a startingmaterial for most PEG activation reactions is typically an end-cappedPEG. An end-capped PEG is one where one or more of the hydroxyl groups,typically located at a terminus of the polymer, is converted into anon-reactive group, such as a methoxy, ethoxy, or benzyloxy group. Mostcommonly used is methoxyPEG, abbreviated as mPEG. End-capped PEGs suchas mPEG are generally preferred, since such end-capped PEGs aretypically more resistant to cross-linking and aggregation. Thestructures of two commonly employed end-capped PEG alcohols, mPEG andmonobenzyl PEG (otherwise known as bPEG), are shown below,

wherein n typically ranges from about 10 to about 2,000.

In one specific example of a polymer reagent for use in drug delivery,U.S. Pat. No. 6,436,386 describes PEG-based, hydroxyapatite-targetingpolymers that can be used to selectively target bone surfaces within apatient for delivery of therapeutic agents to the bone site. In thismanner, the polymeric reagent provides both targeted delivery of theactive portion of the molecule to the tissue of interest and increasedcirculation time.

Despite many successes, conjugation of a polymer to an active agent isoften challenging. For example, it is known that attaching a relativelylong poly(ethylene glycol) molecule to an active agent typically impartsgreater water solubility than attaching a shorter poly(ethylene glycol)molecule. One of the drawbacks of some conjugates bearing polymermoieties, however, is the possibility that such conjugates may besubstantially inactive in vivo. It has been hypothesized that theseconjugates are substantially inactive due to the length of the polymerchain, which effectively “wraps” itself around the entire active agent,thereby limiting access to ligands required for pharmacologic activity.

As a result, there is an ongoing need in the art for polymer reagentssuitable for conjugation to drug moieties for drug deliveryapplications, particularly polymer reagents that have the molecularweight necessary to provide a for a conjugate that has the desirable invivo circulation time, but which also exhibits timely clearance from thebody. It would be particularly beneficial for such polymer reagents toalso provide the ability to target a particular site of the body, suchas hydroxyapatite surfaces. The present invention addresses this andother needs in the art.

SUMMARY OF THE INVENTION

The present invention provides hydroxyapatite-targeting, multiarmpolymer reagents suitable for reaction with biologically active agentsto form conjugates, the polymeric reagents comprising one or morepolymer chains and a plurality of hydroxyapatite-targeting moietieslocated at the terminus of one or more of the polymer chains. Themultiarm polymers are optionally divided or separated by one or moredegradable linkages into polymer segments having a molecular weightsuitable for renal clearance. The polymeric reagents of the inventioncan have a substantially linear structure, although branched or multiarmstructures are contemplated as well. The invention is suited forapplications in which use of a high molecular weight polymer is desired,such as a total polymer number average molecular weight of at leastabout 30,000 Da for linear polymers and 20,000 Da for multiarm polymers.Each structure includes one or more linkages capable of degradation invivo. The use of multiple hydroxyapatite-targeting moieties on eachpolymer molecule enhances the ability of the polymer reagent toselectively target and bind to hydroxyapatite surfaces, which in turn,can increase the concentration of biologically active moiety deliveredto the bone site.

In one embodiment, the invention provides a hydroxyapatite-targeting,multiarm polymer having the structure:

wherein:

A is —(X³)_(d)-(L³)_(e)-(X⁴)_(f)-POLY²-Z² or—(X³)_(d)-(L³)_(e)-(X⁴)_(f)—Z²

each POLY¹ and POLY², which may be the same or different, is awater-soluble, non-peptidic polymer;

each X¹, X², X³, and X⁴, which may be the same or different, is a spacermoiety;

each L¹, L², and L³, which may be the same or different, are linkages;

each Z¹, which may be the same or different, is Z² or ahydroxyapatite-targeting moiety or a multiarm structure comprising 2 toabout 10 hydroxyapatite-targeting moieties and optionally including atleast one water-soluble, non-peptidic polymer, with the proviso that,when b is zero, at least one Z¹ has a multiarm structure comprising oneor more polymer arms and with the proviso that at least one Z¹ is ahydroxyapatite-targeting moiety;

Z² is a functional group, optionally attached to POLY² through a spacer;

each a, b, c, d, e, and f, which may be the same or different, is eitherzero or one;

R is a monomeric or oligomeric multiarm core molecule derived from amolecule comprising at least p+1 sites available for attachment; and

p is an integer in the range of 2-32.

In certain embodiments, each of POLY¹ and POLY² have a number averagemolecular weight satisfying one or more of the following: less thanabout 22,000 Da; less than about 15,000 Da; and less than about 8,000Da. Exemplary polymers for POLY¹ and POLY² include poly(alkyleneglycols), poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(acrylic acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),and copolymers, terpolymers, or mixtures thereof. Examples of thehydroxyapatite-targeting moiety include tetracycline, calcein,bisphosphonates, polyaspartic acid, polyglutamic acid, andaminophosphosugars.

Certain embodiments of the polymer reagents of the invention include atleast one hydrolytically or enzymatically cleavable linkage, such as inthe linkages at the L¹, L², or L³ position. The polymer chains, such asPOLY¹ and POLY², can have a segmented structure comprising two to aboutfive water-soluble, non-peptidic polymer segments attached throughlinkages. For example, one or both of POLY¹ and POLY² can have astructure according to the formula -POLY-L-POLY-, wherein each POLY is awater-soluble, non-peptidic polymer and L is a linkage, the linkageoptionally being degradable.

The terminal Z¹ moiety can have a multiarm structure, such as any of thefollowing structures:

wherein each m is 1-350, Me is methyl, and each Z is ahydroxyapatite-targeting moiety.

The core moiety, R, can be derived from a polyol with the structureR¹(OH)_(p), wherein R is a branched hydrocarbon, optionally includingone or more ether linkages, and p is at least 3. Exemplary polyolsinclude glycerol, pentaerythritol, sugar-derived alcohols, and oligomersor polymers thereof. Alternatively, R can be derived from disulfides,peptides, oligomers or polymers thereof, and combinations thereof. Incertain embodiments, R is derived from a di-peptide or tri-peptidecomprising at least one lysine residue.

Exemplary polymer reagents of the invention include the followingpolymer structures:

wherein n is 1-350.

In another aspect, the invention provides a hydroxyapatite-targeting,multiarm polymer conjugate comprising the reaction product of thepolymer reagent of the invention with a biologically active agent, andhaving the structure:

wherein all previous variables of Formula (Ia) apply to Formula (Ib) andfurther wherein B is —(X³)_(d)-(L³)_(e)—(X⁴)_(f)-POLY²-L⁴-Drug or—(X³)_(d)-(L³)_(e)—(X⁴)_(f)-L⁴-Drug, Drug is a residue of a biologicallyactive moiety, L⁴ is a linkage resulting from reaction of Z² with afunctional group on the biologically active moiety, and Z³ is L⁵-Drug ora hydroxyapatite-targeting moiety, wherein L⁵ is a linkage resultingfrom reaction of Z¹, where Z¹ is a functional group, with a functionalgroup on the biologically active moiety, with the proviso that at leastone Z³ is a hydroxyapatite-targeting moiety.

The Drug is a residue of a biologically active moiety, which can be, forexample, growth factors, antibiotics, chemotherapeutic agents, oranalgesics. Exemplary growth factors include fibroblast growth factors,platelet-derived growth factors, bone morphogenic proteins, osteogenicproteins, transforming growth factors, LIM mineralization proteins,osteoid-inducing factors, angiogenins, endothelins; growthdifferentiation factors, ADMP-1, endothelins, hepatocyte growth factorand keratinocyte growth factor, heparin-binding growth factors, hedgehogproteins, interleukins, colony-stimulating factors, epithelial growthfactors, insulin-like growth factors, cytokines, osteopontin, andosteonectin.

Although multiarm structures are most preferred, in another aspect, theinvention provides a heterobifunctional, substantially linear,hydroxyapatite-targeting polymer having the structure:Z—(X¹)_(a)-L¹-(X²)_(b)-[POLY¹-(X³)_(c)-L²-(X⁴)_(d)]_(m)-POLY²-(X⁵)_(e)—Ywherein:

each POLY¹ and POLY², which may be the same or different, is awater-soluble, non-peptidic polymer;

each X¹, X², X³, X⁴, and X⁵, which may be the same or different, is aspacer moiety;

L¹ is a linkage;

each L² is a hydrolytically or enzymatically cleavable linkage selectedfrom the group consisting of carbamate and amide;

Z is a hydroxyapatite-targeting moiety;

Y is a functional group;

each a, b, c, d, and e, which may be the same or different, is eitherzero or one; and

m is an integer in the range of 1-10.

In addition to polymer reagents and conjugates made therefrom, theinvention includes methods of making such reagents and conjugates, aswell as therapeutic methods of using biologically active conjugates ofpolymer reagents of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to the particularpolymers, synthetic techniques, active agents, and the like as such mayvary. It is also to be understood that the terminology used herein isfor describing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to a “polymer” includesa single polymer as well as two or more of the same or differentpolymers, reference to a “conjugate” refers to a single conjugate aswell as two or more of the same or different conjugates, reference to an“excipient” includes a single excipient as well as two or more of thesame or different excipients, and the like.

I. DEFINITIONS

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” are used hereinto mean any water-soluble poly(ethylene oxide). Typically, PEGs for usein the present invention will comprise one of the two followingstructures: “—O(CH₂CH₂O)_(n)—” or “—CH₂CH₂O(CH₂CH₂O)_(n)—CH₂CH₂—,” wheren is 3 to 3000, and the terminal groups and architecture of the overallPEG may vary. “PEG” means a polymer that contains a majority, that is tosay, greater than 50%, of subunits that are —CH₂CH₂O—.

One commonly employed PEG is end-capped PEG. When PEG is defined as“—O(CH₂CH₂O)_(n)—,” the end-capping group is generally acarbon-containing group typically comprised of 1-20 carbons and ispreferably alkyl (e.g., methyl, ethyl or propyl) although saturated andunsaturated forms thereof, as well as aryl, heteroaryl, cyclo,heterocyclo, and substituted forms of any of the foregoing are alsoenvisioned. When PEG is defined as “—CH₂CH₂O(CH₂CH₂O)_(n)—CH₂CH₂—,” theend-capping group is generally a carbon-containing group typicallycomprised of 1-20 carbon atoms and an oxygen atom that is covalentlybonded to the group and is available for covalently bonding to oneterminus of the PEG. In this case, the group is typically, alkoxy (e.g.,methoxy, ethoxy or benzyloxy) and with respect to the carbon-containinggroup can optionally be saturated and unsaturated, as well as aryl,heteroaryl, cyclo, heterocyclo, and substituted forms of any of theforegoing. The other (“non-end-capped”) terminus is a typicallyhydroxyl, amine or an activated group that can be subjected to furtherchemical modification when PEG is defined as“—CH₂CH₂O(CH₂CH₂O)_(n)—CH₂CH₂—.” In addition, the end-capping group canalso be a silane.

Specific PEG forms for use in the invention include PEGs having avariety of molecular weights, structures or geometries (e.g., branched,linear, multiarm, and the like), to be described in greater detailbelow.

The end-capping group can also advantageously comprise a detectablelabel. When the polymer has an end-capping group comprising a detectablelabel, the amount or location of the polymer and/or the moiety (e.g.,active agent) of interest to which the polymer is coupled can bedetermined by using a suitable detector. Such labels include, withoutlimitation, fluorescers, chemiluminescers, moieties used in enzymelabeling, colorimetric (e.g., dyes), metal ions, radioactive moieties,and the like.

The polymers used in the methods described herein are typicallypolydisperse (i.e., number average molecular weight and weight averagemolecular weight of the polymers are not equal). The polymers preparedin accordance with the methods described herein, however, possess lowpolydispersity values—expressed as a ratio of weight average molecularweight (Mw) to number average molecular weight (Mn), (Mw/Mn)—generallyless than about 1.3, preferably less than about 1.2, more preferablyless than about 1.15, still more preferably less than about 1.05, yetstill most preferably less than about 1.04, and most preferably lessthan about 1.03. It is noted that the polydispersity of a multiarm PEGcould be much higher than the polydispersity of the polymer arms used tocreate the multiarm PEG.

As used herein, the term “ionizable functional group” and variationsthereof is a functional group that may gain or lose a proton byinteraction with another ionizable species of functional group inaqueous or other polar media. Ionizable functional groups include, butare not limited to, amine, carboxylic acids, aldehyde hydrates, ketonehydrates, amides, hydrazines, thiols, phenols, oximes, dithiopyridines,and vinylpyridines.

As used herein, the term “carboxylic acid” is a moiety having a

functional group also represented as a “—COOH” or —C(O)OH], as well asmoieties that are derivatives of a carboxylic acid, such derivativesincluding, for example, protected carboxylic acids. Thus, unless thecontext clearly dictates otherwise, the term carboxylic acid includesnot only the acid form, but corresponding esters and protected forms aswell. Reference is made to Greene et al., “PROTECTIVE GROUPS IN ORGANICSYNTHESIS” 3^(rd) Edition, John Wiley and Sons, Inc., New York, 1999.

“Activated carboxylic acid” means a functional derivative of acarboxylic acid that is more reactive than the parent carboxylic acid,in particular, with respect to nucleophilic acyl substitution. Activatedcarboxylic acids include but are not limited to acid halides (such asacid chlorides), anhydrides, amides and esters.

The term “reactive” or “activated”, when used in conjunction with aparticular functional group, refers to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “nonreactive” or “inert” group).

The terms “protected” or “protecting group” or “protective group” referto the presence of a moiety (i.e., the protecting group) that preventsor blocks reaction of a particular chemically reactive functional groupin a molecule under certain reaction conditions. The protecting groupwill vary depending upon the type of chemically reactive group beingprotected as well as the reaction conditions to be employed and thepresence of additional reactive or protecting groups in the molecule, ifany. Protecting groups known in the art can be found in Greene et al.,supra.

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof.

The term “spacer” or “spacer moiety” is used herein to refer to an atomor a collection of atoms optionally used to link interconnectingmoieties such as a terminus of a water-soluble polymer and a functionalgroup. The spacer moieties of the invention may be hydrolytically stableor may include a physiologically hydrolyzable or enzymaticallydegradable linkage.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includeethyl, propyl, butyl, pentyl, 2-methylbutyl, 2-ethylpropyl, and thelike. As used herein, “alkyl” includes cycloalkyl when alkyl can includethree or more carbon atoms.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, iso-butyl, tert-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃-C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl (e.g., 0-2substituted phenyl); substituted phenyl; and the like.

“Substituted aryl” is aryl having one or more non-interfering groups asa substituent. For substitutions on a phenyl ring, the substituents maybe in any orientation (i.e., ortho, meta, or para).

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy,benzyloxy, etc.), more preferably C₁-C₈ alkyl.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof. Heteroaryl rings mayalso be fused with one or more cyclic hydrocarbon, heterocyclic, aryl,or heteroaryl rings.

“Electrophile” refers to an ion or atom or collection of atoms, that maybe ionic, having an electrophilic center, i.e., a center that iselectron seeking or capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or collection of atoms, that maybe ionic, having a nucleophilic center, i.e., a center that is seekingan electrophilic center or capable of reacting with an electrophile.

A linkage that is “cleavable in vivo” refers to linkages capable ofbeing cleaved while in circulation in vivo by a hydrolytic process, anenzymatic process, a chemical process, or a combination of suchprocesses. In other words, linkages that are cleavable in vivo are thoselinkages that can break apart under physiological conditions (i.e., atabout pH 7 to 7.5 and temperature of about 37° C. in the presence ofserum or other body fluids). The degradation half-life of the linkagecan vary, but is typically in the range of about 0.1 to about 10 daysunder physiologic conditions.

A “hydrolytically cleavable” or “hydrolyzable” or “hydrolyticallydegradable” bond is a relatively weak bond that reacts with water (i.e.,is hydrolyzed) under physiological conditions. The tendency of a bond tohydrolyze in water will depend not only on the general type of linkageconnecting two central atoms but also on the substituents attached tothese central atoms. Appropriate hydrolytically unstable or weaklinkages include, but are not limited to, carboxylate ester, phosphateester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines,orthoesters, and oligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes under physiological conditions. Theenzymatic degradation process may also include a hydrolysis reaction.Enzymatically degradable linkages can include certain amide (—C(O)—NH—)and urethane (—O—C(O)—NH—) linkages, especially when in a proximatearrangement with other groups of atoms that may provide eitheractivation for degradation or additional sites needed for attraction ofan enzyme. For example, a urethane in proximate location with certainamides, e.g. —O—C(O)—NH—CHY—C(O)—NH—Y′, where Y is H, alkyl, substitutedalkyl (e.g., arylalkyl, hydroxylalkyl, thioalkyl, etc.), or aryl, and Y′is alkyl or substituted alkyl, are enzymatically degradable. As definedherein, “urethane” linkages are inclusive of linkages having the abovestructure.

A “chemically degradable” linkage as used herein is a linkage thatdegrades through chemical reaction under physiologic conditions in vivo.For example, disulfide (—S—S—) bonds can be degraded in vivo throughchemical reaction with glutathione.

A “hydrolytically stable” or “non-degradable” linkage or bond refers toa chemical bond, typically a covalent bond, that is substantially stablein water, meaning it does not undergo hydrolytic or enzymatic cleavageunder physiological conditions to any appreciable extent over anextended period of time. Examples of hydrolytically stable linkagesinclude but are not limited to the following: carbon-carbon bonds (e.g.,in aliphatic chains), ethers, and the like. Generally, a hydrolyticallystable linkage is one that exhibits a rate of hydrolysis of less thanabout 1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Multifunctional” or “multisubstituted” in the context of a polymer orpolyol means a polymer or polyol having two or more functional groupscontained therein, where the functional groups may be the same ordifferent. Multifunctional polymers or polyols of the invention willtypically contain a number of functional groups satisfying one or moreof the following ranges: from about 2-100 functional groups, from 2-50functional groups, from 2-25 functional groups, from 2-15 functionalgroups, from 2 to 10 functional groups. Thus, the number of functionalgroups in the polymer backbone or polyol can be any one of 2, 3, 4, 5,6, 7, 8, 9 or 10 functional groups.

A “difunctional” or “disubstituted” polymer or polyol means a polymer orpolyol having two functional groups contained therein, either the same(i.e., homodifunctional) or different (i.e., heterodifunctional).

A “monofunctional” or “monosubstituted” polymer means a polymer having asingle functional group contained therein (e.g., an mPEG based polymer).

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

Unless otherwise noted, molecular weight is expressed herein as numberaverage molecular weight (M_(n)), which is defined as

$\frac{\sum{NiMi}}{\sum{Ni}},$wherein Ni is the number of polymer molecules (or the number of moles ofthose molecules) having molecular weight Mi.

Each of the terms “drug,” “biologically active molecule,” “biologicallyactive moiety,” “active agent” and “biologically active agent”, whenused herein, means any substance which can affect any physical orbiochemical property of a biological organism, including but not limitedto viruses, bacteria, fungi, plants, animals, and humans. In particular,as used herein, biologically active molecules include any substanceintended for diagnosis, cure, mitigation, treatment, or prevention ofdisease in humans or other animals, or to otherwise enhance physical ormental well-being of humans or animals. Examples of biologically activemolecules include, but are not limited to, peptides, proteins, enzymes,small molecule drugs, dyes, lipids, nucleosides, oligonucleotides,polynucleotides, nucleic acids, cells, viruses, liposomes,microparticles and micelles. Classes of biologically active agents thatare suitable for use with the invention include, but are not limited to,antibiotics, fungicides, anti-viral agents, anti-inflammatory agents,anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones,growth factors, steroidal agents, and the like.

As used herein, “non-peptidic” refers to a polymer backbonesubstantially free of peptide linkages. However, the polymer backbonemay include a minor number of peptide linkages spaced along the lengthof the backbone, such as, for example, no more than about 1 peptidelinkage per about 50 monomer units.

The term “conjugate” is intended to refer to the entity formed as aresult of covalent attachment of a molecule, e.g., a biologically activemolecule, to a reactive polymer molecule, preferably a poly(ethyleneglycol) bearing one or more reactive groups.

II. HYDROXYAPATITE-TARGETING POLYMERS AND CONJUGATES MADE THEREFROM

In one aspect, the present invention provides polymeric reagents, andconjugates with biologically active agents made using the polymericreagents, characterized by the presence of a plurality ofhydroxyapatite-targeting moieties. The use of a plurality ofhydroxyapatite-targeting moiety in a single polymer structure canenhance binding of the polymer to a bone surface, which couldpotentially increase the residence time of a drug molecule attached tothe polymer structure at the bone site.

A generalized structure of a multiarm polymer of the invention is shownbelow, which includes bone-targeting moieties and a reactive handle thatcan be used to conjugate a therapeutic agent. The number ofbone-targeting moieties (BTM) may vary extensively from two to more thantwenty, depending on the binding efficiency of the particular BTM. Thepolymer molecular weight can be adjusted as desired to provide maximumefficiency in the various roles that the polymer plays, e.g.,circulation time, solubility, protection of the drug, and carrier of theBTMs.

wherein each POLY and the optional POLY₂ is a water-soluble,non-peptidic polymer, BTM is a bone-targeting moiety (used hereininterchangeably with hydroxyapatite-targeting moiety), and L₁, L₂, andL₃ are linkages. Conjugation of the “reactive handle” with a therapeuticagent, such as a drug that may target bone cancer cells, makes thepolymer-bound drug a targeting drug for bone cells. In application, thedrug could be injected directly into the cancerous portion of the bonefor action.

The reactive handle is preferably an ionizable functional group that canbe utilized in manipulation and purification of the molecule. Exemplaryionizable functional groups include amine and carboxylic acid groupssuch as alkanoic acids having a carbon length (including the carbonylcarbon) of 1 to about 25 carbon atoms (e.g., carboxymethyl, propanoicacid, and butanoic acid). Examples of other suitable functional groupsinclude aldehyde hydrate, ketone hydrate, amide, hydrazine, hydrazide,thiol, sulfonic acid, amidate, hydroxylamine, phenol, oxime,dithiopyridine, vinylpyridine, 2-substituted-1,3-oxazoline,2-substituted 1,3-(4H)-dihydrooxazines, 2-substituted-1,3-thiazoline,and 2-substituted 1,3-(4H)-dihydrothiazines.

An alternate generalized structure of a multiarm polymer of theinvention is shown below, which includes a single bone-targeting moietyand several sites containing therapeutic agents. Again, the number BTMsmay vary extensively from one to several, depending on the bindingefficiency of the particular BTM, but the preferred number of BTM unitsis low. The polymer molecular weight can be adjusted as desired toprovide maximum efficiency in the various roles that the polymer plays,e.g., circulation time, solubility, and carrier of the BTMs. The drugmoieties in this application would be low molecular weight units thatalternatively could be attached to the polymer by a degradable linkagethat would allow delivery of the drug at the targeted site.

The polymer reagents may also include one or more cleavable ordegradable linkages that degrade in vivo. The degradable linkage orlinkages are spaced along the polymer chain or within a central coremolecule such that each segment of polymeric reagent that is releasedupon degradation of the linkage in vivo has a molecular weight that doesnot impede renal clearance of the segment. The polymeric reagents of thepresent invention are particularly advantageous in that they can be usedto prepare conjugates where both a relatively high polymer molecularweight is desired along with substantially complete elimination of thepolymer from the body. For example, the total polymer number averagemolecular weight for the polymeric reagent (and the conjugate preparedtherefrom) is typically at least about 30,000 Da, such as a molecularweight of about 30,000 to about 150,000 Da (e.g., total molecularweights of about 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, 50,000 Da,55,000 Da, 60,000 Da, 65,000 Da, 70,000 Da, and the like). The numberaverage molecular weight of each polymer segment released upondegradation of the degradable linkages is preferably less than or equalto about 22,000 Da, more preferably less than or equal to about 20,000Da, even more preferably less than or equal to about 15,000 Da, and mostpreferably less than or equal to about 8,000 Da. In some embodiments,the polymer segments have a molecular weight of no more than about 5,000Da, or no more than about 2,500 Da. The number of polymer segmentsresulting from cleavage of the degradable linkages can vary from 2 toabout 40, but is generally in the range of 2 to about 10 (e.g., 2, 3, 4,5, 6, 7, 8, 9, or 10 polymer segments).

The structural configuration of the polymeric reagents (and theconjugates prepared therefrom) of the invention can vary. Although lesspreferred, the polymeric reagents can have a substantially linear form.Preferred embodiments of the polymeric reagent have a “multiarm”configuration comprising two or more (preferably three or more) polymerarms extending from a common multifunctional core molecule, such as apolyol or peptide. Preferred embodiments of the polymers of theinvention are not in the form of a hydrogel, meaning the polymericreagents (and the conjugates prepared therefrom) are not crosslinked toa substantial degree with other polymers in a water-swellable matrix.

The degradable linkages within the polymeric reagents (and theconjugates prepared therefrom) can vary. It is preferable to usedegradable linkages cleavable in vivo, and having a half-life of betweenabout 0.1 and about 10 days under physiological conditions (i.e., at apH of 7-7.5 and a temperature of about 37° C.). The rate of degradationof a linkage can be measured by analytical determination of liberatedpolymer segments using gel permeation chromatography (“GPC”). Althoughthe polymeric reagents of the invention can include one or morecarbonate groups as a degradable linkage, it is preferable for thepolymeric reagents to comprise at least one degradable linkage that doesnot include a carbonate group, and polymeric reagents without anycarbonate groups are contemplated.

Exemplary degradable linkages include, but are not limited to, esterlinkages; carbonate linkages; carbamates; imides; disulfides; di-, tri-,or tetrapeptides; imine linkages resulting, for example, from reactionof an amine and an aldehyde (see, e.g., Ouchi et al. (1997) PolymerPreprints 38(1):582-3, which is incorporated herein by reference);phosphate ester linkages formed, for example, by reacting an alcoholwith a phosphate group; hydrazone linkages which are typically formed byreaction of a hydrazide and an aldehyde; acetal linkages that aretypically formed by reaction between an aldehyde and an alcohol; orthoester linkages that are, for example, formed by reaction between aformate and an alcohol; and oligonucleotide linkages formed by, forexample, a phosphoramidite group, e.g., at the end of a polymer, and a5′ hydroxyl group of an oligonucleotide.

Amide and urethane bonds are generally considered stable groups forbinding PEGs to proteins such as interferon, e.g., K. R. Reddy, M. W.Modi and S. Pedder (2002) Adv. Drug Delivery Rev. 54:571-586. Somecleavage of these stable groups, however, may occur in vivo. Forexample, in a PEG interferon (marketed under the “PEGASYS®” brand), upto 30% of the PEG associated with the conjugate is cleared by cleavageof a urethane bond (see M. W. Modi, J. S. Fulton, D. K. Buckmann, T. L.Wright, D. J. Moore (2000) “Clearance of Pegylated (40 kDa) interferonalpha-2a (PEGASYS®) is Primarily Hepatic, Hepatology 32: 371A). Themechanism for the overall clearance of the conjugate is fairly slow andtakes several days.

With respect to amide bounds, there are special cases where amide bonds,such as those found in peptide linkages, are susceptible to enzymaticcleavage. Suzawa et al. (2000) Bioorg. Med. Chem. 8(8):2175-84) foundthat a poly(ethylene glycol) bound L-alanine-valine di-peptide linkagecleaved in the presence of the model enzyme thermolysin. Additionalexamples of peptide linkages (e.g., di-peptide or tri-peptide linkages)that may find use in the present invention can be found in U.S. Pat.Nos. 5,286,637 and 6,103,236; Goff and Carroll (1990) Bioconjugate Chem.1:381-386); and Huang et al. (1998) Bioconjugate Chem. 9:612-617). Thus,in certain embodiments, the degradable linkage(s) contained within thepolymeric reagents (and the conjugates formed therefrom) can includeamide or urethane linkages.

Esters, though more susceptible than amides and urethanes to hydrolyticcleavage, are also readily cleaved by enzymatic processes, thus makingesters especially labile linkages in vivo. Esters are more resistant toenzymatic cleavage if they have groups in the vicinity of the functionalgroup that sterically block the approach of an enzyme. Hence, includingthis type of sterically hindered ester function may cause an ester groupto be an attractive linker for applications where it is desirable forthe polymer to break down hydrolytically or enzymatically in a few hoursto a few days.

The groups that best facilitate stability through steric hindrance aregroups (e.g., alkyl groups) located at the position alpha to thecarbonyl carbon of the ester, as is the case with the twoester-containing polymers below (wherein “POLY” is a water-soluble,non-peptidic polymer). In selecting a structure to present a sterichindrance to enzymatic cleavage, it is preferred to not include a groupthat has an electron withdrawing effect on the carbonyl group. While notwishing to be bound by theory, such electron withdrawing groups wouldtend to accelerate acid- or base-catalyzed hydrolysis.

Steric hindrance in the alkyl portion (e.g., the portion or atomsproximal to the oxygen atom, which, in turn, is attached to the carbonylcarbon) of the ester may also slow enzymatic cleavage of esters. Thus,when steric hindrance is desired to influence the rate of enzymaticcleavage, it is contemplated to add steric hindrance at the alpha and/orbeta positions relative to the carbonyl carbon and/or the oxygen atom,which, in turn, is attached to the carbonyl carbon of the ester group.It is important, however, to add a combination of steric crowding andelectron donation so as to facilitate electrophilic cleavage of theester by a S_(N)1 pathway. Further, it is important to not make thealkyl portion such a good leaving group, by substitution of electronwithdrawing groups, that base catalyzed hydrolysis is favorable. Abalance can be achieved by the introduction of mild steric retardationat the alpha and beta positions of the oxygen atom, which, in turn, isattached to the carbonyl carbon of the ester group, as shown in thestructure below.

wherein L is a spacer moiety or a linkage resulting from reaction ofPOLY with an ester-containing moiety and POLY is a water-soluble,non-peptidic polymer.

Thus, preferred steric hindering groups include alkyl groups (e.g.,C1-C10 alkyl groups) or aryl groups (e.g., C6-C10 aryl groups)positioned adjacent to the carbonyl carbon and/or adjacent to the oxygenatom attached to the carbonyl group of the ester (i.e., at the alpha orbeta positions), and most preferably adjacent to the carbonyl carbon.

It is possible to determine whether any given proposed group is suitedfor providing the desired steric hindrance by preparing the polymericreagent with the proposed group. Following formation of the conjugatefrom the proposed polymeric reagent, the conjugate is subsequentlyadministered the conjugate to a patient or added to a suitable model.Following administration to the patient (or addition to the suitablemodel), the degradative rate for each degradable linkage within theconjugate can be determined by, for example, taking a blood sample (oraliquot of liquid from the suitable model) and identifying degradativecomponents of the conjugate through chromatographic techniques. Theproposed group is suited for providing the desired steric hindrance ifthe overall degradation rate falls within a desired range and/or isimproved over a control polymeric reagent tested under the sameconditions.

The water-soluble, non-peptidic polymers (e.g., POLY¹, POLY², and soforth) that make up part of the polymeric reagents of the presentinvention should be non-toxic and biocompatible, meaning that thepolymer is capable of coexistence with living tissues or organismswithout causing harm. It is to be understood that the polymer can be anyof a number of water-soluble, non-peptidic polymers. Preferably,poly(ethylene glycol) (i.e., PEG) is the polymer used to form thepolymeric reagents described herein. Examples of other suitable polymersinclude, but are not limited to, other poly(alkylene glycols),copolymers of ethylene glycol and propylene glycol, poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(acrylic acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), such as described in U.S. Pat. No.5,629,384, which is incorporated by reference herein in its entirety,and copolymers, terpolymers, and mixtures thereof. Different polymerscan be incorporated into the same polymer backbone. Any combination ofwater soluble and non-peptidic polymers is encompassed within thepresent invention. Each polymer segment (e.g., each POLY¹ or POLY²) canalso comprise two or more polymer segments connected by cleavable orstable linkages.

The polymers can be in substantially linear form or a multiarm orbranched form, such as the branched PEG molecules set forth in U.S. Pat.No. 5,932,462, which is incorporated by reference herein in itsentirety. Generally speaking, a multiarmed or branched polymer possessestwo or more polymer “arms” extending from a central branch point. Forexample, an exemplary branched PEG polymer has the structure:

wherein PEG₁ and PEG₂ are PEG polymers in any of the forms or geometriesdescribed herein, and which can be the same or different, and L′ is ahydrolytically stable linkage. An exemplary branched PEG of Formula Ihas the structure:

wherein: poly_(a) and poly_(b) are PEG backbones, such as hydroxypoly(ethylene glycol); R″ is a nonreactive moiety, such as H, methyl ora PEG backbone; and P and Q are nonreactive linkages. In a preferredembodiment, the branched PEG polymer is hydroxy poly(ethylene glycol)disubstituted lysine.

The branched PEG structure of Formula IV can be attached to a thirdoligomer or polymer chain as shown below:

wherein PEG₃ is a third PEG oligomer or polymer chain, which can be thesame or different from PEG₁ and PEG₂.

In another embodiment, the branched PEG used in the invention has thestructure:

wherein each POLY is a water-soluble, non-peptidic polymeric oroligomeric segment (e.g., a PEG segment), and each Z is a capping group,a functional group, or a bone-targeting group.

As evidenced in the exemplary polymeric structures below, the polymericreagents of the invention will typically include one or more functionalgroups suitable for reaction with a complementary functional group on abiologically active agent in order to form a covalent linkage (which canoptionally be cleavable in vivo) between the polymeric reagent and theactive agent. Examples of suitable functional groups include hydroxyl,active ester (e.g., N-hydroxysuccinimidyl ester and 1-benzotriazolylester), active carbonate (e.g., N-hydroxysuccinimidyl carbonate,1-benzotriazolyl carbonate, and p-nitrophenyl carbonate), acetal,aldehyde having a carbon length of 1 to 25 carbons (e.g., acetaldehyde,propionaldehyde, and butyraldehyde), aldehyde hydrate, alkenyl,acrylate, methacrylate, acrylamide, active sulfone, amine, hydrazide,thiol, alkanoic acids having a carbon length (including the carbonylcarbon) of 1 to about 25 carbon atoms (e.g., carboxylic acid,carboxymethyl, propanoic acid, and butanoic acid), acid halide,isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine,vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate,tosylate, and tresylate. Exemplary functional groups are discussed inthe following references: N-succinimidyl carbonate (see e.g., U.S. Pat.Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al. Makromol.Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J. 19:1177 (1983)),hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301 (1978)),succinimidyl propionate and succinimidyl butanoate (see, e.g., Olson etal. in Poly(ethylene glycol) Chemistry & Biological Applications, pp170-181, Harris & Zalipsky Eds., ACS, Washington, D.C., 1997; see alsoU.S. Pat. No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowskiet al. Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al.,Makromol. Chem. 180:1381 (1979), succinimidyl ester (see, e.g., U.S.Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem.94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991),oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem.131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)),p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem.Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech.,27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem.Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714),maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romaniet al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan,Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g.,Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461). All of the above references are incorporatedherein by reference.

In certain embodiments, the capping group, functional group, orhydroxyapatite-targeting group (a “Z” moiety such as Z¹, Z², Z³, and soforth) of the polymeric reagents (and the conjugates formed therefrom)will have a multiarm structure. For example, the “Z” moiety can be amultiarm reactive structure comprising 2 to about 10 functional groups(e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 functional groups). Exemplarymultiarm groups include those having the structure

wherein each Z, which may be the same or different, is a functionalgroup, a capping group, or a hydroxyapatite targeting group, optionallyincluding a spacer moiety, and m is an integer in the range of 1 toabout 350, preferably 1 to about 10, more preferably 1 to about 4.

The polymeric reagents (and the conjugates formed therefrom) may includeone or more spacer moieties (an “X” moiety such as X¹, X², X³, X⁴, X⁵,X⁶, X⁷, X⁸, X⁹, and so forth), particularly located on either side ofdegradable or stable linkages resulting from reaction of two polymerspecies or a polymer and a biologically active agent. Exemplary spacermoieties include —C(O)O—, —OC(O)—, —CH₂—C(O)O—, —CH₂—OC(O)—,—C(O)O—CH₂—, —OC(O)—CH₂—, —C(O)—, —C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—,—C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—,—CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,—CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—C—O—C(O)—NH—[CH₂]_(h)—(OCH₂CH₂)_(j)—,—NH—C(O)—O—[CH₂]_(h)—(OCH₂CH₂)_(j)—, bivalent cycloalkyl group, —O—,—S—, an amino acid, a di- or tri-peptide, —N(R⁶)—, and combinations oftwo or more of any of the foregoing, wherein R⁶ is H or an organicradical selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl andsubstituted aryl, (h) is zero to six, and (j) is zero to 20. Otherspecific spacer moieties have the following structures:—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, and—O—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, wherein the subscript values followingeach methylene indicate the number of methylenes contained in thestructure, e.g., (CH₂)₁₋₆ means that the structure can contain 1, 2, 3,4, 5 or 6 methylenes. The spacer moiety may also comprise an ethyleneoxide oligomer/polymer chain comprising 1 to 25 ethylene oxide monomerunits [i.e., —(CH₂CH₂O)₁₋₂₅], either in addition to the above-describedspacer moieties or in lieu thereof. When used in addition to anotherspacer moiety, the ethylene oxide oligomer chain can occur before orafter the spacer moiety, and optionally in between any two atoms of aspacer moiety comprised of two or more atoms.

Preferred biologically active agents for use in the conjugates of theinvention include active agents having relatively low water solubility,such as certain proteins, peptides, and small molecule drugs.Particularly preferred biologically active agents include those that areintended to have a biological effect on bone tissue within the patient,such as growth factors, antibiotics, chemotherapeutic agents, oranalgesics. Exemplary growth factors include fibroblast growth factors,platelet-derived growth factors, bone morphogenic proteins, osteogenicproteins, transforming growth factors, LIM mineralization proteins,osteoid-inducing factors, angiogenins, endothelins; growthdifferentiation factors, ADMP-1, endothelins, hepatocyte growth factorand keratinocyte growth factor, heparin-binding growth factors, hedgehogproteins, interleukins, colony-stimulating factors, epithelial growthfactors, insulin-like growth factors, cytokines, osteopontin, andosteonectin.

Other examples of relatively hydrophobic active agents that can becovalently attached to polymeric reagents of the invention include, butare not limited to, abietic acid, aceglatone, acenaphthene,acenocournarol, acetohexamide, acetomeroctol, acetoxolone,acetyldigitoxins, acetylene dibromide, acetylene dichloride,acetylsalicylic acid, alantolactone, aldrin, alexitol sodium, allethrin,allylestrenol, allylsulfide, alprazolam, aluminum bis(acetylsalicylate),ambucetamide, aminochlothenoxazin, aminoglutethimide, amyl chloride,androstenediol, anethole trithone, anilazine, anthralin, Antimycin A,aplasmomycin, arsenoacetic acid, asiaticoside, asternizole, aurodox,aurothioglycanide, 8-azaguanine, azobenzene, baicalein, Balsam Peru,Balsam Tolu, barban, baxtrobin, bendazac, bendazol, bendroflumethiazide,benomyl, benzathine, benzestrol, benzodepa, benzoxiquinone,benzphetamine, benzthiazide, benzyl benzoate, benzyl cinnamate,bibrocathol, bifenox, binapacryl, bioresmethrin, bisabolol, bisacodyl,bis(chlorophenoxy)methane, bismuth iodosubgallate, bismuth subgallate,bismuth tannate, Bisphenol A, bithionol, bornyl, bromoisovalerate,bornyl chloride, bornyl isovalerate, bornyl salicylate, brodifacoum,bromethalin, broxyquinoline, bufexamac, butamirate, butethal,buthiobate, butylated hydroxyanisole, butylated hydroxytoluene, calciumiodostearate, calcium saccharate, calcium stearate, capobenic acid,captan, carbamazepine, carbocloral, carbophenothin, carboquone,carotene, carvacrol, cephaeline, cephalin, chaulmoogric acid, chenodiol,chitin, chlordane, chlorfenac, chlorfenethol, chlorothalonil,chlorotrianisene, chlorprothixene, chlorquinaldol, chromonar,cilostazol, cinchonidine, citral, clinofibrate, clofaziminc, clofibrate,cloflucarban, clonitrate, clopidol, clorindione, cloxazolam, coroxon,corticosterone, cournachlor, coumaphos, coumithoate cresyl acetate,crimidine, crufomate, cuprobam, cyamemazine, cyclandelate, cyclarbamatecymarin, cyclosporin A, cypermethril, dapsone, defosfamide,deltamethrin, deoxycorticocosterone acetate, desoximetasone,dextromoramide, diacetazoto, dialifor, diathymosulfone, decapthon,dichlofluani, dichlorophen, dichlorphenamide, dicofol, dicryl,dicumarol, dienestrol, diethylstilbestrol, difenamizole,dihydrocodeinone enol acetate, dihydroergotamine, dihydromorphine,dihydrotachysterol, dimestrol, dimethisterone, dioxathion, diphenane,N-(1,2-diphenylethyl)nicotinamide, 3,4-di-[1-methyl6-nitro-3-indolyl]-1H-pyrrole-2,5-dione (MNIPD), dipyrocetyl,disulfamide, dithianone, doxenitoin, drazoxolon, durapatite, edifenphos,emodin, enfenamic acid, erbon, ergocorninine, erythrityl tetranitrate,erythromycin stearate, estriol, ethaverine, ethisterone, ethylbiscournacetate, ethylhydrocupreine, ethyl menthane carboxamide,eugenol, euprocin, exalamide, febarbamate, fenalamide, fenbendazole,fenipentol, fenitrothion, fenofibrate, fenquizone, fenthion, feprazone,flilpin, filixic acid, floctafenine, fluanisone, flumequine, fluocortinbutyl, fluoxymesterone, fluorothyl, flutazolam, fumagillin,5-furftiryl-5-isopropylbarbituric acid, fusaftmgine; glafenine,glucagon, glutethimide, glybuthiazole, griseofulvin, guaiacol carbonate,guaiacol phosphate; halcinonide, hematoporphyrin, hexachlorophene,hexestrol, hexetidine, hexobarbital, hydrochlorothiazide, hydrocodone,ibuproxam, idebenone, indomethacin, inositol niacinate, iobenzamic acid,iocetamic acid, iodipamide, iomeglamic acid, ipodate, isometheptene,isonoxin, 2-isovalerylindane-1,3-dione, josamycin, 11-ketoprogesterone,laurocapram, 3-O-lauroylpyridoxol diacetate, lidocaine, lindane,linolenic acid, liothyronine, lucensomycin, mancozeb, mandelic acid,isoamyl ester, mazindol, mebendazole, mebhydroline, mebiquine,melarsoprol, melphalan, menadione, menthyl valerate, mephenoxalone,mephentermine, mephenyloin, meprylcaine, mestanolone, mestranol,mesulfen, metergoline, methallatal, methandriol, methaqualone,methylcholanthrene, methylphenidate, 17-methyltestosterone,metipranolol, minaprine, myoral, naftalofos, naftopidil, naphthalene,2-naphthyl lactate, 2-(2-naphthyloxy)ethanol, naphthyl salicylate,naproxen, nealbarbital, nemadectin, niclosamide, nicoclonate,nicomorphine, nifuroquine, nifuroxazide, nitracrine, nitromersol,nogalamycin, nordazepam, norethandrolone, norgestrienone, octaverine,oleandrin, oleic acid, oxazeparn, oxazolam, oxeladin, oxwthazaine,oxycodone, oxymesterone, oxyphenistan acetate, paclitaxel,paraherquamide, parathion, pemoline, pentaerythritol tetranitrate,pentylphenol, perphenazine, phencarbamide, pheniramine,2-phenyl-6-chlorophenol, phenthnethylbarbituric acid, phenyloin,phosalone, O-phthalylsulfathiazole, phylloquinone, picadex, pifamine,piketopfen, piprozolin, pirozadil, pivaloyloxymethyl butyrate,plafibride, plaunotol, polaprezinc, polythiazide, probenecid,progesterone, promegestone, propanidid, propargite, propham, proquazone,protionamide, pyrimethamine, pyrimithate, pyrvinium pamoate, quercetin,quinbolone, quizalofo-ethyl, rafoxanide, rescinnamine, rociverine,ronnel, salen, scarlet red, siccanin, simazine, simetride, simvastatin,sobuzoxane, solan, spironolactone, squalene, stanolone, sucralfate,sulfabenz, sulfaguanole, sulfasalazine, sulfoxide, sulpiride,suxibuzone, talbutal, terguide, testosterone, tetrabromocresol,tetrandrine, thiacetazone, thiocolchicine, thioctic acid, thioquinox,thioridazine, thiram, thymyl N-isoamylcarbamate, tioxidazole, tioxolone,tocopherol, tolciclate, tolnaftate, triclosan, triflusal, triparanol,ursolic acid, valinomycin, verapamil, vinblastine, vitamin A, vitamin D,vitamin E, xenbucin, xylazine, zaltoprofen, and zearalenone.

In one embodiment, the invention provides a hydroxyapatite-targeting,multiarm polymer having the structure:

wherein:

A is —(X³)_(d)-(L₃)_(e)-(X₄)_(f)-POLY²-Z² or—(X³)_(d)-(L³)_(e)-(X⁴)_(f)—Z²

each POLY¹ and POLY², which may be the same or different, is awater-soluble, non-peptidic polymer;

each X¹, X², X³, and X⁴, which may be the same or different, is a spacermoiety;

each L¹, L², and L³, which may be the same or different, are linkages;

each Z¹, which may be the same or different, is Z² or ahydroxyapatite-targeting moiety or a multiarm structure comprising 2 toabout 10 hydroxyapatite-targeting moieties and optionally including atleast one water-soluble, non-peptidic polymer, with the proviso that,when b is zero, at least one Z¹ has a multiarm structure comprising oneor more polymer arms and with the proviso that at least one Z¹ is ahydroxyapatite-targeting moiety;

Z² is a functional group (e.g., an ionizable functional group),optionally attached to POLY² through a spacer;

each a, b, c, d, e, and f, which may be the same or different, is eitherzero or one;

R is a monomeric or oligomeric multiarm core molecule derived from amolecule comprising at least p+1 sites available for attachment; and

p is an integer in the range of 2-32.

In certain embodiments, each of POLY¹ and POLY² have a number averagemolecular weight of less than about 22,000 Da, less than about 15,000Da, or less than about 8,000 Da. Exemplary polymers for POLY¹ and POLY²include poly(alkylene glycols), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(acrylic acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), and copolymers, terpolymers, or mixturesthereof. Examples of the hydroxyapatite-targeting moiety includetetracycline, calcein, bisphosphonates, polyaspartic acid, polyglutamicacid, and aminophosphosugars.

Certain embodiments of the polymer reagents of the invention include atleast one hydrolytically or enzymatically cleavable linkage as notedabove, such as at the L¹, L², or L³ position. The polymer chains, suchas POLY¹ and POLY², can have a segmented structure comprising two toabout five water-soluble, non-peptidic polymer segments attached throughlinkages. For example, one or both of POLY¹ and POLY² can have astructure according to the formula -POLY-L-POLY-, wherein each POLY is awater-soluble, non-peptidic polymer and L is a linkage, which can beenzymatically or hydrolytically cleavable.

In another aspect, the invention provides a hydroxyapatite-targeting,multiarm polymer conjugate comprising the reaction product of thepolymer reagent of the invention with a biologically active agent, andhaving the structure:

wherein B is —(X³)_(d)-(L³)_(e)-(X⁴)_(f)-POLY²-L⁴-Drug or—(X³)_(d)-(L³)_(e)-(X⁴)_(f)-L⁴-Drug, Drug is a residue of a biologicallyactive moiety, L⁴ is a linkage resulting from reaction of Z² with afunctional group on the biologically active moiety, and Z³ is L⁵-Drug ora hydroxyapatite-targeting moiety, wherein L⁵ is a linkage resultingfrom reaction of Z¹, where Z¹ is a functional group, with a functionalgroup on the biologically active moiety, with the proviso that at leastone Z³ is a hydroxyapatite-targeting moiety.

The core molecule, R, can be any monomeric or oligomeric moleculeproviding three or more reactive sites for attachment of polymersegments, and will typically include between 3 and about 32 reactivesites, more preferably between 3 and about 25 reactive sites, and mostpreferably between 3 and about 10 reactive sites (e.g., 3, 4, 5, 6, 7,8, 9, or 10 reactive sites). Note that the number of reactive sites onthe core molecule can be greater than the number of sites actually usedfor attachment to polymer segments (i.e., the number of reactive sitescan be greater than p). The reactive sites comprise terminal functionalgroups available for reaction with functionalized polymeric segments,and may include more than one type of functional group. For instance,certain di- or tri-peptide core molecules will comprise both one or morecarboxylic acid groups and one or more amine groups. As noted above, theR core molecule can be a combination of a polypeptide (e.g., di- ortri-peptide) or disulfide with a polyol to form a multiarm core moleculeto which polymer arms can be attached at the site of the hydroxyl groupsof the polyol and/or at the site of any free reactive groups on thepolypeptide or disulfide. Note that the R core molecule does not have tobe preformed prior to attachment of the polymer arms. Instead, the coremolecule can be created after polymer arms have been attached to one ofthe components that will form the ultimate core molecule. For example,polymer arms can be attached to a polyol molecule prior to attachment oftwo polymer-modified polyol molecules together through a disulfide ordi-peptide linker.

A polyol used as the core molecule comprises a plurality of availablehydroxyl groups. Depending on the desired number of polymer arms, thepolyol will typically comprise 3 to about 25 hydroxyl groups, preferablyabout 3 to about 22 hydroxyl groups, most preferably about 5 to about 12hydroxyl groups. Although the spacing between hydroxyl groups will varyfrom polyol to polyol, there are typically 1 to about 20 atoms, such ascarbon atoms, between each hydroxyl group, preferably 1 to about 5. Theparticular polyol chosen will depend on the desired number of hydroxylgroups needed as attachment sites for the polymer arms. The numberaverage molecular weight of the polyol starting material is typicallybetween about 100 to about 2,000 Da. The polyol typically has a branchedstructure, meaning one or more carbon atoms in the hydrocarbon corestructure of the polyol are covalently attached to three or four atomsselected from carbon atoms and ether-linked oxygen atoms (i.e., oxygenatoms attached to two carbon atoms).

Preferred polyols for use as the core molecule include glycerololigomers or polymers such as hexaglycerol, pentaerythritol andoligomers or polymers thereof (e.g., dipentaerythritol,tripentaerythritol, and tetrapentaerythritol), and sugar-derivedalcohols such as sorbitol, arabanitol, and mannitol. Also, manycommercially available polyols containing ionizable groups, such as2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS),2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol,{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}acetic acid (Tricine),2-[(3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}propyl)amino]-2-(hydroxymethyl)-1,3-propanediol,2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid(TES), 4-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-butanesulfonicacid, and 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediolhydrochloride are appropriate starting materials. Typically, polymericpolyols used in the present invention will comprise no more than about25 monomer units. The structures of dipentaerythritol andtripentaerythritol are provided below along with one of the structurespossible for hexaglycerol.

Hydroxypropyl-β-cyclodextrin, which has 21 available hydroxyl groups, isanother exemplary polyol. Yet another exemplary polyol is ahyperbranched polyglycerol available from Hyperpolymers GmbH ofFreiburg, Germany, which is shown below.

The polyol may include PEG oligomer or polymer segments attached to thepolyol core. The polyol starting material is typically in the form of amixture of products, such as a mixture of polyol oligomers or polymersof different molecular weights or a mixture of ethoxylated polyolstructures of different molecule weight, possibly further comprising aresidual amount of the original polyol monomeric unit, such as glycerol.However, at least one of the polyols in the starting mixture istypically a branched polyol having at least three available hydroxylgroups according to the formula R(OH)_(p), wherein R is a branchedhydrocarbon, optionally including one or more ether linkages, and p isat least 3, typically 3 to about 25, and preferably 3 to about 10.

Polyols having closely-spaced hydroxyl groups are particularly preferredin certain embodiments of the invention, which facilitate use of cyclicacetal or ketal groups as hydroxyl-protecting groups. A spacing of twoor three carbon atoms between hydroxyl groups within the polyolstructure enables the formation of certain preferred heterocyclicprotecting groups. For example, the close spacing between hydroxylgroups of pentaerythritol oligomers or polymers enable the formation ofcyclic acetal or ketal groups using techniques known in the art. Thecyclic acetal or ketal groups can be formed by reacting the polyol withan aldehyde reagent, such as a reagent having the formula R′—CHO,wherein R′ is alkyl, substituted alkyl, aryl, or substituted aryl, or aketone reagent (e.g., cyclohexanone). An exemplary aldehyde reagent isbenzaldehyde.

By placing a majority of the hydroxyl groups of the polyol in aprotected form, the polyol core can be reacted with a reagent comprisingthe ionizable functional group to produce a plurality of productsdifferentiated by the number of ionizable functional groups presenttherein. Typically, the reaction will produce a monofunctionalizedproduct, a difunctionalized product, and residual unreacted polyol. Anion exchange chromatography system can be used to separate each productfraction based on difference in charge, thereby allowing purification ofthe desired monofunctional product. A process for purifying PEG polymerspecies based on charge differences is set forth in U.S. PatentApplication Publication No. 2005/0054816, which is hereby incorporatedby reference in its entirety.

The ion exchange column or columns utilized in the purification processcan be any ion exchange columns conventionally used to separate amixture based on charge (Ion Exchange Chromatography. Principles andMethod. Pharmacia Biotech 1994; “Chromatography: a laboratory handbookof chromatographic and electrophoretic techniques.” Heftman, E (Ed.),Van Nostrand Rheinhold Co., New York, 1975). Each column comprises anion exchange media and a mobile phase or eluent that passes through theion exchange media. Ion exchange columns suitable for use in the presentinvention include POROS® ion exchange media made by Applied Biosystemsand SEPHAROSE® ion exchange media made by Pharmacia.

In certain embodiments, each POLY¹ is a poly(ethylene glycol) polymer,and R is a disulfide linker, a dipeptide, a tripeptide, or atetrapeptide, which means the R moiety will include at least onedisulfide bond (from the disulfide linker) or amide bond (e.g., thelinkage between peptide residues). Preferred R groups include thosecomprising at least one lysine residue. Suitable disulfide linkersinclude various linkers comprising an —S—S— bond and a total of 4-25atoms in chain length, and preferred disulfide molecules have 4-8functional groups available for attachment of polymer segments. Incertain embodiments, each POLY¹ and POLY² is a branched poly(ethyleneglycol) polymer.

Polymeric reagent can comprise R moieties derived from a disulfidemolecule having the structure:

In further embodiments, each POLY¹ comprises a poly(ethylene glycol)polymer, and R is comprises at least one peptide residue. The R moietymay further comprise a disulfide bond. In certain embodiments, Rcomprises at least two lysine residues linked by amide linkages to alinker selected from the group consisting of an aliphatic carbon chain,an aliphatic carbon chain comprising a disulfide bond, and apoly(ethylene glycol) oligomer (e.g., an oligomer having from 1-25monomer units).

In still further embodiments, each POLY¹ comprises a poly(ethyleneglycol) polymer and R comprises a non-peptidic moiety comprising atleast one disulfide bond and at least two amide bonds. By “non-peptidic”is meant that the R molecule does not include a peptide residue (i.e.,the amide and disulfide bonds are not part of a peptide molecule). Inthis manner, R core molecules can be used that mimic peptidic moleculesin structure due to inclusion of amide linkages, but which are nottechnically peptidic in nature.

Although multiarm structures are most preferred, in another aspect, theinvention provides a heterobifunctional, substantially linear,hydroxyapatite-targeting polymer having the structure:Z—(X¹)_(a)-L¹-(X²)_(b)-[POLY¹-(X³)_(c)-L²-(X⁴)_(d)]-POLY²-(X⁵)_(e)—Ywherein:

each POLY¹ and POLY², which may be the same or different, is awater-soluble, non-peptidic polymer;

each X¹, X², X³, X⁴, and X⁵, which may be the same or different, is aspacer moiety;

L¹ is a linkage;

each L² is a hydrolytically or enzymatically cleavable linkage selectedfrom the group consisting of carbamate and amide;

Z is a hydroxyapatite-targeting moiety;

Y is a functional group;

each a, b, c, d, and e, which may be the same or different, is eitherzero or one; and

m is an integer in the range of 1-10.

Exemplary polymer reagents of the invention include the followingpolymer structures:

Example 1 provides an exemplary synthesis route for the first structurenoted above. The second structure above is based upon a “PEG2” moleculewith a glycerol core, two remote bisphosphonate groups on the PEG chaintermini, and a site for drug attachment through the butanoic acidfunctional group. To construct this molecule, one can use abenzyl-capped PEG (b-PEG), rather than a typical methoxy-capped PEG(m-PEG). Then, in an appropriate processing step, the benzyl groups canbe removed by hydrogenolysis and, through a series of steps, thebisphosphonate functions can be added. Example 2 illustrates a possiblesynthetic route to molecules of this type. Example 3 provides anexemplary synthesis for the trilysine-based polymer reagent noted above.

The method of forming a hydroxyapatite-targeting multiarm polymer of theinvention can vary. Reaction Scheme 1 below illustrates one method ofconstructing a six-arm polyol, based on pentaerythritol, having an esteras the ionizable reactive arm (protected carboxylic acid). In thisexample, the linker L₃ is a multifunctional linker, such as lysine.

Reaction Scheme 2 below shows how the above generalized structure ofReaction Scheme 1 is deprotected (by hydrogenolysis of the benzyl ester)and then activated for subsequent reaction with an amine-terminaltherapeutic agent.

Reaction Scheme 3 below shows a method of attaching a bone-targetingmoiety to the termini of a polymer having a lysine core and a carboxylicacid group as the ionizable reactive arm. As shown, the trityl groupsare subjected to hydrogenolysis, followed by esterification of the acidgroup to form a protected acid, and then reaction of the termini with abone-targeting agent, which in this case is AHPDP (a derivative of3-amino-1-hydroxypropane-1,1-diphosphonic acid). The resulting polymericspecies, in Reaction Scheme 3 below, can be subjected to hydrogenolysisto remove the benzyl ester group. Then, reaction with DCC/NHS, followingconditions similar to those used in U.S. Pat. No. 6,436,386 B1, which isincorporated by reference, can convert this bone-targeting polymer intoan active ester reagent (NHS ester) that can be used to conjugatetherapeutic agents through an available amine group on the therapeuticagent, e.g., an N-terminal lysine.

As indicated above, certain homo-oligomers of amino acids having a freecarboxyl group, such as polyaspartic acid or polyglutamic acid, have agood binding capacity for hydroxyapatite. Therefore, an alternatestructure for a bone-targeting polymer can be prepared using this typeof polypeptide group as a pendant group. Shown in the scheme below(Reaction Scheme 4) is a way that a commercially availableheterobifunctional PEG derivative can be reacted with a polyasparticacid, which is known in the art. The product, which still has both theamine and terminal carboxylic acids blocked, can be further manipulatedto deblock the amine group. This intermediate can be used directly informing a multiarm reagent (see Reaction Scheme 5, for example) or itcan be further manipulated to form an active ester, which can beconverted into a different multiarm reagent, which is not shown.

Reaction Scheme 5 below illustrates the formation of amine- andacid-protected intermediates used in preparing a polypeptide-containingbone-targeting multiarm polymeric reagent.

In Reaction Scheme 6 below, the methyl ester intermediate from ReactionScheme 5 is deprotected by base hydrolysis, which will not affect thet-butyl esters. The resulting carboxylic acid is then esterified to anactive ester with dicyclohexylcarbodiimide and N-hydroxysuccinimide.Reaction with an amine-bearing reagent for forming a maleimide gives thepolymeric reagent bearing a maleimide group and still having thecarboxylic acid esters on the polypeptide moieties. A mild acidhydrolysis will remove the protecting groups giving a reagent that isreactive toward thiol-containing therapeutic agents.

In Reaction Scheme 7 below, the conjugation with the reagent above isillustrated with a therapeutic agent, which happens to be a polypeptide,bearing a thiol group.

The invention includes polymer reagents and conjugates made therefromthat are designed to act as a prodrug and release the biologicallyactive moiety at the bone site. In this case, a degradable functionalgroup is added at the site where conjugation to the drug occurs.Degradable functional groups of the phenolate or FMOC type can readilybe incorporated into this type of molecule and thus are hereby includedas a feature of this class of reagent.

In Reaction Scheme 8 below is shown an example of a polymeric reagentcontaining a tripeptide link that can be enzymatically cleaved in vivoto allow for the polymer to clear as smaller fragments. Usingcommercially available trilysine as a core tripeptide, the free aminegroups are reacted with a benzyl protected active carbonate PEG(b-PEG-BTC). This leads to formation of a four-arm polymer with theremote termini protected with a removable benzyl group. Note that thepolymer has a carboxylic acid group that can be used to purify thepolymer and also to activate for connection to a drug molecule.

Completion of a series of processing steps to the product of ReactionScheme 8 allows addition of the bone-targeting functionality and thedrug leading to the four-arm bisphosphonate set forth below.

In an alternate process, a four-arm structure can be derived from use ofthe monoblocked (ester) disulfide linked dipeptide Lys-Cys, i.e. shownbelow.

This small molecule can be elaborated into the four-arm bisphosphonatepolymeric drug delivery conjugate illustrated below. The advantage ofusing the segmented polymeric species resides in the ability of thesepolymers to break down, through enzyme or intracellular chemical action,into smaller, linear fragments that will clear from the body morequickly than stable multiarm molecules like those based on certainpolyols.

Following pathways known in the art, the molecule above could cleave atthe bonding sites designated by the dotted lines. The single dottedlines are sites where enzymatic or intracellular chemical cleavage islikely and the double dotted lines represent sites where depegylationmay occur through a chemical process that involves a neighboring amidegroup (Guiotto et al, Biorg. Med. Chem. 2004, 12, 5031-5037). Disulfidebonds in water soluble polymer-bound drug molecules are known to undergocleavage in serum or in the cell by agents like glutathione (Zalipsky etat Bioconj. Chem. 1999, 10, 703-707; U.S. Pat. No. 6,342,244B1; US PatAppl. 2005/0170508A1; Huang et al, Bioconj Chem. 1998, 9, 612-617).Enzymatic cleavage of peptide bonds has also been reported with watersoluble polymers (Ulbrich et al Makromol. Chem. 1986, 187, 1131-1144;Suzawa et al, J. Controlled Rel. 2000, 69, 27-41; Suzawa et al U.S. Pat.No. 6,103,236).

The use of segmented polymers having bisphosphonate bone-targetinggroups may be important when a high molecular weight polymer is desiredto enhance delivery and retention of the conjugate until bone targetinghas been achieved. Then, segment cleavage would allow clearance of thepolymer fragments as they would all be of lower molecular weight andgenerally linear polymers with linkers.

In addition to the polymer reagents, conjugates made therefrom, andmethods of synthesis described above, the invention further includesmethods of using the polymer conjugates therapeutically to treat variousconditions and disease states that would benefit from the targeteddelivery of a biologically active agent to the surface of bone.Exemplary conditions to be treated include bone cancer, infections ofbone tissue, age-induced degradation of bone tissue, bone defects causedby trauma, and the like. The choice of administration route,biologically active moiety, and dosage range can be readily determinedby the clinician and will vary based on numerous factors including thecondition to be treated, the condition of the patient, the severity ofthe injury or disease, and the like.

All articles, books, patents, patent publications and other publicationsreferenced herein are hereby incorporated by reference in theirentireties.

EXPERIMENTAL

It is to be understood that while the invention has been described inconjunction with certain preferred specific embodiments thereof, theforegoing description as well as the example that follows are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.For example, in certain applications, it may be desirable to utilize apolymeric reagent according to any of the above formulas wherein alllinkages therein are stable rather than degradable.

All PEG reagents referred to in the appended example are commerciallyavailable unless otherwise indicated, e.g., from Nektar Therapeutics,Huntsville, Ala. All ¹HNMR data was generated by a 300 or 400 MHz NMRspectrometer manufactured by Bruker. High Performance LiquidChromatography (HPLC) was performed using Agilent 1100 HPLC system(Agilent), gel permeation or ion exchange column, aqueous phosphatebuffer as a mobile phase, and refractive index (RI) detector.

Example 1 I. PEG(5,000 Da)-α-Hydroxy-ω-Butanoic Acid, Methyl Ester

To a solution of PEG(5,000)-α-hydroxy-ω-butanoic acid (70 g, 0.0140moles)(Nektar Therapeutics) in anhydrous methanol (400 ml) was addedconcentrated sulfuric acid (8.0 ml) followed by stirring of the mixturefor 3 h at room temperature. NaHCO₃ (8% aqueous solution) was added toadjust the pH of the mixture to 7.0. Methanol was distilled off underreduced pressure and the product was extracted with CH₂Cl₂ (2×350 ml).The extract was dried (MgSO₄) and the solvent was distilled off underreduced pressure. Yield 60 g.

NMR (d₆-DMSO): 1.72 ppm (m, —CH₂ CH₂COO—, 2H), 2.34 ppm (t, —CH₂COO—,2H), 3.51 ppm (s, PEG backbone), 4.57 ppm (t, —OH, 1H), 3.58 ppm (s,CH₃O—, 3H).

II. PEG(5,000 Da)-α-Succinimidyl Carbonate-ω-Butanoic Acid, Methyl Ester

To a solution of PEG(5,000 Da)-α-hydroxy-ω-butanoic acid, methyl ester(60 g, 0.0120 moles) in acetonitrile (300 ml), pyridine (1.60 ml) anddisuccinimidyl carbonate (3.92 g) were added and the reaction mixturewas stirred overnight at room temperature under argon atmosphere. Nextthe mixture was filtered and solvent was evaporated to dryness. Thecrude product was dissolved in methylene chloride and precipitated withisopropyl alcohol. The wet product was dried under reduced pressure.Yield 57 g.

NMR (d₆-DMSO): 1.72 ppm (m, —CH₂CH₂COO—, 2H), 2.34 ppm (t, —CH₂COO—,2H), 2.81 ppm (s, —CH₂CH₂— (succinimide), 4H), 3.51 ppm (s, PEGbackbone), 3.58 ppm (s, —CH₃O—, 3H), 4.45 ppm (m, —CH₂—O(C═O)—, 2H).

III. PEG(5,000 Da)-α-AHPDP-ω-Butanoic Acid

To a solution of PEG(5,000 Da)-α-succinimidyl carbonate-ω-butanoic acid,methyl ester (40 g, 0.0080 moles) in acetonitrile (400 ml),3-amino-1-hydroxypropane-1,1-diphosphonic acid, ditetrabutylammoniumsalt (AHPDP-2Bu₄N) (6.2 g) and triethylamine (2.4 ml) were added and thereaction mixture was stirred overnight at room temperature under argonatmosphere. Next solvent was evaporated to dryness. The crude productwas dissolved in DI water (400 ml) and the pH of the solution wasadjusted to 12.0 with 1M sodium hydroxide. The solution was stirred 2 hkeeping the pH at 12 by periodic addition of 1M sodium hydroxide then itwas filtered through Amberlite IR 120 (plus) column (200 ml). From thefiltrate, water was distilled off under reduced pressure. The wetproduct was dissolved in methylene chloride (600 ml) then the solventwas distilled off. Finally the product was dried under reduced pressure.Yield 35 g.

NMR (d₆-DMSO): 1.72 ppm (m, —CH₂ CH₂COO—, 2H), 2.02 ppm (m,—CH₂-(AHPDP), 2H), 2.34 ppm (t, —CH₂COO—, 2H), 3.51 ppm (s, PEGbackbone), 4.03 ppm (m, —CH₂—O(C═O)—, 2H), 7.11 ppm (t, —(C═O)NH—, 1H).

IV. PEG(5,000)-α-AHPDP-ω-Butanoic Acid, N-Hydroxysuccinimide Ester

To a solution of PEG(5,000 Da)-α-AHPDP-ω-butanoic acid (30 g, 0.0060equivalents) in anhydrous methylene chloride (300 ml),N-hydroxysuccinimide (0.83 g, 0.0072 moles) was added following by1,3-dicyclohexylcarbodiimide (1.0 M solution in methylene chloride, 7.2ml, 0.0072 moles). The reaction mixture was stirred overnight at roomtemperature under an argon atmosphere. Next the mixture was filtered andsolvent was evaporated to dryness. The crude product was dissolved inmethylene chloride and precipitated with isopropyl alcohol. Finally theproduct was dried under reduced pressure. Yield 27 g.

NMR (d₆-DMSO): 1.84 ppm (m, —CH₂ CH₂COO—, 2H), 2.02 ppm (m,—CH₂-(AHPDP), 2H), 2.71 ppm (t, —CH₂COO—, 2H), 2.81 ppm (s, —CH₂CH₂—(succinimide), 4H), 3.51 ppm (s, PEG backbone), 4.03 ppm (m,—CH₂—O(C═O)—, 2H), 7.11 ppm (t, —(C═O)—NH—, 1H).

V. PEG(5,000)-α-AHPDP-ω-Butyraldehyde Diethyl Acetal

To a solution of PEG(5,000 Da)-α-AHPDP-ω-butanoic acid,N-hydroxysuccinimide ester (25 g, 0.0050 equivalents) in anhydrousmethylene chloride (250 ml), tetra(ethyleneglycol)-α-amino-ω-butyraldehyde, diethyl acetal (Nektar Therapeutics;2.0 g, 0.0059 moles) was added following by triethylamine (1.70 ml). Thereaction mixture was stirred overnight at room temperature under argonatmosphere. Next the mixture was filtered and solvent was evaporated todryness. The crude product was dissolved in methylene chloride andprecipitated with isopropyl alcohol. Finally the product was dried underreduced pressure. Yield 22 g.

NMR (d₆-DMSO): 1.10 ppm (t, CH₃—C, 6H), 1.51 ppm (m, C—CH₂—CH₂—,butyraldehyde, 4H), 1.72 ppm (m, —CH₂ CH₂COO—, 2H), 2.02 ppm (m,—CH₂-(AHPDP), 2H), 2.10 ppm (t, —CH₂COO—, 2H), 3.51 ppm (s, PEGbackbone), 4.03 ppm (m, —CH₂—O(C═O)—, 2H), 4.46 ppm (t, —CH—, acetal,1H), 7.11 ppm (t, —(C═O)—NH—, 1H).

VI. PEG(5,000)-α-AHPDP-ω-Butyraldehyde

PEG(5,000)-α-AHPDP-ω-butyraldehyde diethyl acetal (20 g) was dissolvedin 300 ml water and the pH of the solution was adjusted to 2.5 withdiluted phosphoric acid. The solution was stirred 3 hours at roomtemperature. Next 0.5M sodium hydroxide was used to adjust the pH of thesolution to 7. The product was extracted with methylene chloride andprecipitated with diethyl ether. Finally the product was dried underreduced pressure. Yield 17.5 g.

NMR (d₆-DMSO): 1.75 ppm (m, —CH₂ CH₂CHO, 2H and —CH₂ CH₂COO—, 2H), 2.02ppm (m, —CH₂-(AHPDP), 2H), 2.10 ppm (t, —CH₂COO—, 2H), 2.44 ppm (dt,—CH₇ CHO, 2H), 3.51 ppm (s, PEG backbone), 4.03 ppm (m, —CH₂O(C═O)—,2H), 7.11 ppm (t, —(C═O)—NH—, 1H), 9.66 ppm (t, —CHO, 1H).

Example 2 I. Preparation of a Glycerol-Based Precursor Molecule

A solution of cis-1,3-O-Benzylideneglycerol (7.2 g, 0.040 moles)(Sigma-Aldrich Corporation, St. Louis, Mo.) in toluene (100 ml) wasazetropically dried by distilling off toluene. The dried compound wasdissolved in anhydrous toluene (100 ml) and 1.0M solution of potassiumtert-butoxide in tert-butanol (60 ml, 0.060 moles) and1-(3-bromopropyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (14.0 g,0.0558 moles) were added and the mixture was stirred overnight at 100°C. under argon atmosphere. The mixture was filtered and the solvent wasdistilled off under reduced pressure giving 15.7 g of solid product(Compound 2). NMR (d₆-DMSO): 0.74 ppm (s, 3H), 1.61 ppm (m, 4H), 1.88ppm (m, 2H), 3.44 ppm (t, 2H), 3.81 ppm (s, 6H), 4.05 ppm (m, 4H), 5.55ppm (s, 1H), 7.37 ppm (m, 5H).

Schematically, the reaction is represented as follows:

Hydrolysis of Compound 2

Compound 2 (15.0 g) was dissolved in a mixture of acetonitrile (150 ml)and distilled water (35 ml). Next, a 10% solution of H₃PO₄ was added toadjust the pH to 4.5. The mixture was stirred for 1 hour at pH=4.5. NaCl(2 g) was added and the pH was adjusted to 7.5. The product wasextracted with CH₂Cl₂ (600 and 150 ml).

The extract was dried (MgSO₄) and the solvent was distilled off underreduced pressure to give a solid product (Compound 3). The yield wasdetermined to be 14.2 g.

NMR (d₆-DMSO): 0.78 ppm (s, 3H), 1.79 ppm (m, 2H), 2.41 ppm (t, 2H),3.25 ppm (m, 6H), 3.49 ppm (t, 2H), 4.05 ppm (m, 4H), 4.48 ppm (t, 3H),5.56 ppm (s, 1H), 7.37 ppm (m, 5H).

Schematically, the reaction is represented as follows:

Compound 3 (14.2 g) was dissolved in a mixture of acetonitrile (80 ml)and distilled water (80 ml). Next, a 6% solution of NaOH was added toadjust the pH to 12.5. The solution was stirred for 5.5 hours at pHranging from 12.3-12.8, which was maintained by periodical additions ofa 6% solution of NaOH. NaCl (5 g) was added and the pH was adjusted to7.5 with 5% H₃PO₄. The non-acidic impurities were extracted with CH₂Cl₂(two treatments, a first using 300 ml and a second using 200 ml). The pHof the solution was adjusted to 3.0 with H₃PO₄ and the product wasextracted with CH₂Cl₂ (two treatments, a first using 200 ml and a secondusing 100 ml).

The extract was dried (MgSO₄) and the solvent was distilled off underreduced pressure. The resulting product (Compound 4) had a yield of 8.7g.

NMR (d₆-DMSO): 1.76 ppm (m, 2H), 2.31 ppm (t, 2H), 3.46 ppm (t, 2H),4.05 ppm (m, 4H), 5.56 ppm (s, 1H), 7.37 ppm (m, 5H).

Schematically, the reaction is represented as follows:

Compound 4 (8.0 g) was dissolved in anhydrous methanol (120 ml) and upondissolution, concentrated H₂SO₄ (1.6 ml) was added. The solution wasstirred for 4 hours at room temperature. NaHCO₃ (8% solution) was addedto adjust the pH of the mixture to 7.5. The product was extracted withCH₂Cl₂ (two treatments, each using 100 ml).

The extract was dried (MgSO₄) and volatile compounds were distilled offunder reduced pressure (0.05 mm Hg) at 60° C. The resulting product(Compound 1) had a yield of 4.8 g.

NMR (d₆-DMSO): 1.72 ppm (m, 2H), 2.37 ppm (t, 2H), 3.20 ppm (m, 1H),3.42 ppm (bm, 4H), 3.49 ppm (t, 2H), 3.59 ppm (s, 3H), 4.46 ppm (t, 2H).

Schematically, the reaction is represented as follows:

II. Preparation of “HO-PEG2_((20K))-Butanoic Acid, N-HydroxysuccinimideEster”

(wherein HO-PEG_(10K) designates a PEG having a molecular weight of10,000 Daltons) “HO-PEG2_((20K))-Butanoic Acid, N-HydroxysuccinimideEster”

Activation of the Hydroxyl Groups in the Precursor Molecule

Compound 1 (2.0 g, 0.0208 equivalents) was dissolved in anhydrousacetonitrile (50 ml) and anhydrous pyridine (2.2 ml, 0.272 mole) andN,N-disuccinimidyl carbonate (5.86 g, 0.0229 mole, DSC) were added. Thesolution was stirred overnight at room temperature under argonatmosphere. Next, the mixture was filtered and the solvent was distilledoff. The crude product was dissolved in CH₂Cl₂ (50 ml) and washed with a5% H₃PO₄ solution. The solution was then dried (MgSO₄), and the solventwas distilled off. The resulting product (Compound 5) had a yield of 2.8g.

NMR (d₆-DMSO): 1.76 ppm (m, 2H), 2.35 ppm (t, 2H), 2.82 ppm (s, 8H),3.56 ppm (t, 2H), 3.58 ppm (s, 3H), 3.96 ppm (m, 1H), 4.37 ppm (m, 2H),4.52 ppm (m, 2H).

Schematically, the reaction is represented as follows:

Coupling the Activated Precursor with an Amine-Containing Water-SolublePolymer

To a mixture of Benzyloxy-PEG_((5K))-amine (BzO-PEG_((5K))-amine) (20 g,0.0040 mole) (Nektar Therapeutics, Huntsville, Ala.), methylene chloride(200 ml), and triethylamine (1.4 ml), Compound 5 (0.901 g, 0.0038equivalents) was added. The mixture was stirred overnight at roomtemperature under argon atmosphere. Next, the solvent was distilled offunder reduced pressure.

Schematically, the reaction is represented as follows:

Deprotecting Step and Chromatographic Purification ofBzO-PEG2_((20K))-Butanoic Acid

The obtained Compound 6 (herein referred to as BzO-PEG2_((20K))-butanoicacid, methyl ester) was dissolved in 400 ml of distilled water and thepH of the solution was adjusted to 12.2 with a 0.5M NaOH solution. Thesolution was stirred for 3 hours at a pH in a range of 12.0-12.2. Next,NaCl (20 g) was added and the pH was adjusted to 3.0 with a 5% H₃PO₄solution. The product was extracted with a CH₂Cl₂ (150 ml×2). Theextract was dried (MgSO₄), and the solvent was distilled off underreduced pressure giving 19 g of solid product. The product was purifiedby ion exchange chromatography as described in U.S. Pat. No. 5,932,462giving 14.5 g of 100% pure product.

NMR (d₆-DMSO): 1.72 ppm (q, —CH₂ CH₂COO—, 2H) 2.24 ppm (t, —CH₂COO—,2H), 3.12 ppm (q, —CH₂ NH—, 4H), 3.51 ppm (s, PEG backbone), 3.99 ppm(m, —CH₂ O(C═O)NH—, 4H), 4.49 ppm (s, —CH₂— (benzyl), 4H), 7.19 ppm (t,—(C═O)NH—, 2H), 7.33 ppm (m, C₆H₅—, 10H).

Schematically, the reaction is represented as follows:

Preparation of BzO-PEG2_((10K))-Butanoic Acid, N-HydroxysuccinimideEster

BzO-PEG2_((10K))-butanoic acid (14.5 g, 0.00145 mole) (prepared asdescribed above) was dissolved in anhydrous dichloromethane (150 ml) andN-hydroxysuccinimide (0.179 g, 0.00156 mole) and 1,3-dicyclocarbodiimide(0.336 g, 0.00163 mole) were added. The mixture was stirred overnight atroom temperature under argon atmosphere. Next, part of the solvent wasdistilled off under reduced pressure and the product was precipitatedwith isopropyl alcohol at room temperature and dried under vacuum giving14.0 g of white powder.

NMR (d₆-DMSO): 1.81 ppm (q, —CH₂ CH₂COO—, 2H), 2.70 ppm (t, —CH₂COO—,2H), 2.81 ppm (s, —CH₂CH₂— (succinimide), 4H), 3.12 ppm (q, —CH₂ NH—,4H), 3.51 ppm (s, PEG backbone), 3.99 ppm (m, —CH₂ (C═O)NH—, 4H), 4.49ppm (t, —CH₂-(benzyl), 4H), 7.22 ppm (t, —(C═O)NH—, 2H), 7.33 ppm (m,C₆H₅—, 10H).

Preparation of HO-PEG2_((10K))-Butanoic Acid, N-Hydroxysuccinimide Ester

BzO-PEG2_((10K))butanoic acid, N-hydroxysuccinimide ester (12.3 g,0.00123 mole) (prepared as described above) was dissolved in anhydrousethyl alcohol (240 ml) and palladium hydroxide on the active carbon (20wt. % of Pd, water content 50%; 0.7 g) was added and the reactionmixture was hydrogenated under 40 psi of hydrogen overnight. Next themixture was filtered and the solvent was distilled off under reducedpressure. The product was precipitated with isopropyl alcohol at roomtemperature and dried under vacuum giving 11.5 g of white powder.

NMR (d₆-DMSO): 1.81 ppm (q, —CH₂ CH₂COO—, 2H), 2.70 ppm (t, —CH₂COO—,2H), 2.81 ppm (s, —CH₂CH₂—, succinimide, 4H), 3.12 ppm (q, —CH₂ NH—,4H), 3.51 ppm (s, PEG backbone), 3.99 ppm (m, —CH₂ ONH(C═O), 4H), 4.57ppm (t, —OH, 2H), 7.22 ppm (t, —(C═O)—NH—, 2H).

Preparation of HO-PEG2_((20K))-Butyraldehyde, Diethyl Acetal

HO—(CH₂CH₂O)₄—CH₂(CH₂)₂—CH(OCH₂CH₂)₂

A mixture of tetra(ethylene glycol) (97.1 g, 0.500 moles) and toluene(200 ml) was azeotropically dried by distilling off toluene underreduced pressure (rotary evaporator). The dried tetra(ethylene glycol)was dissolved in anhydrous toluene (180 ml) and 1.0 M solution ofpotassium tert-butoxide in tert-butanol (120.0 ml, 0.120 moles) and4-chlorobutyraldehyde diethyl acetal (18.1 g, 0.100 moles) (Alfa Aesar,Ward Hill, Mass.) were added. The mixture was stirred at 95-100° C.overnight under argon atmosphere. After cooling to room temperature, themixture was filtered and the solvents were distilled off under reducedpressure. The crude product was dissolved in 1000 ml deionized water andthe resulting solution was filtered through active carbon. Sodiumchloride (100 g) was added and the product was extracted withdichloromethane (250, 200, and 150 ml). The extract was dried (overMgSO₄) and the solvent was distilled off under reduced pressure (byrotary evaporation).

The crude product was dissolved in 300 ml 10% phosphate buffer (pH=7.5)and impurities were extracted with ethyl acetate (2×50 ml). Theresulting product was extracted with dichloromethane (200, 150, and 100ml). The extract was dried (over MgSO₄) and the solvent was distilledoff under reduced pressure (by rotary evaporation). Yield: 20.3 g.

NMR (d₆-DMSO): 1.10 ppm (t, CH₃—C—) 1.51 ppm (m, C—CH₂—CH₂—), 3.49 ppm(bm, —OCH₂CH₂O—), 4.46 ppm (t, —CH, acetal), 4.58 ppm (t, —OH). Purity:˜100% (no signs of unreacted starting materials).

Preparation of Tetra(Ethylene Glycol)-α-Mesylate-ω-Butyraldehyde,Diethyl Acetal

CH₃—S(O)₂—O—(CH₂CH₂O)₄CH₂(CH₂)₂—CH(OCH₂CH₂)₂

A mixture of tetra(ethylene glycol)mono-butyraldehyde, diethyl acetal(12.5 g, 0.037 moles) and toluene (120 ml) was azeotropically dried bydistilling off toluene under reduced pressure (rotary evaporator). Thedried tetra(ethylene glycol)mono-butyraldehyde, diethyl acetal wasdissolved in anhydrous toluene (100 ml). To the solution was added 20 mlof anhydrous dichloromethane and 5.7 ml of triethylamine (0.041 moles).Then 4.5 g of methanesulfonyl chloride (0.039 moles) was added dropwise.The solution was stirred at room temperature under a nitrogen atmosphereovernight. Next sodium carbonate (5 g) was added, the mixture wasstirred for one hour. The solution was then filtered and solvents weredistilled off under reduced pressure (rotary evaporator).

NMR (d₆-DMSO): 1.10 ppm (t, CH₃—C—) 1.51 ppm (m, C—CH₂—CH₂—), 3.17 ppm(s, CH₃— methanesulfonate), 3.49 ppm (bm, —OCH₂CH₂O—), 4.30 ppm (m,—CH₂— methanesulfonate), 4.46 ppm (t, —CH, acetal). Purity: ˜100%.

Tetra(Ethylene Glycol)-α-Amino-ω-Butyraldehyde, Diethyl Acetal

H₂N—(CH₂CH₂O)₄CH₂(CH₂)₂—CH(OCH₂CH₂)₂

A mixture of tetra(ethylene glycol)-α-mesylate-ω-butyraldehyde, diethylacetal (14.0 g), concentrated ammonium hydroxide (650 ml), and ethylalcohol (60 ml) was stirred for 42 hours at room temperature. Next, allvolatile materials were distilled off under reduced pressure. The crudeproduct was dissolved in 150 ml deionized water and the pH of thesolution was adjusted to 12 with 1.0 M NaOH. The product was extractedwith dichloromethane (3×100 ml). The extract was dried (MgSO₄) and thesolvent was distilled off under reduced pressure (rotary evaporator).Yield 10.6 g.

NMR (D₂O): 1.09 ppm (t, CH₃—C—) 1.56 ppm (m, C—CH₂—CH₂—), 2.69 ppm (t,CH₂—N), 3.56 ppm (bm, —OCH₂CH₂O—), 4.56 ppm (t, —CH, acetal). Purity:˜100%.

HO-PEG2(10 KDa)-Butyraldehyde, Diethyl Acetal

To a solution of HO-PEG2_((10K))-butanoic acid, N-hydroxysuccinimideester (10.6 g, 0.00106 moles) in methylene chloride (100 ml),tetra(ethylene glycol)-α-amino-ω-butyraldehyde, diethyl acetal (0.40 g,0.00118 moles) and triethylamine (0.037 ml) were added and the reactionmixture was stirred overnight at room temperature under an argonatmosphere. The solvent was evaporated to dryness using a rotaryevaporator. The crude product was dissolved in methylene chloride andprecipitated with isopropyl alcohol. The wet product was dried underreduced pressure. Yield 10.5 g.

NMR (d₆-DMSO): 1.10 ppm (t, CH₃ CH₂—, 6H), 1.51 ppm (m,—CH₂CH₂-(butyraldehyde), 4H), 1.67 ppm (m, —CH₂ CH₂COO—, 2H), 2.12 ppm(t, —CH₂COO—, 2H), 3.12 ppm (q, —CH₂ NH—, 4H), 3.51 ppm (s, PEGbackbone), 3.99 ppm (m, —CH₂—O(C═O)—, 4H), 4.46 ppm (t, 1H, acetal).4.57 ppm (t, —OH, 2H), 7.22 ppm (t, —(C═O)NH—, 2H), 7.82 ppm (t,—(C═O)NH—, 1H). Substitution: ˜100%.

BTC-PEG2(10 KDa)-Butyraldehyde, Diethyl Acetal

To a solution of HO-PEG2_((10K))-butyraldehyde, diethyl acetal (10.5 g,0.00105 moles) in anhydrous acetonitrile (140 ml), pyridine (0.68 ml)and dibenzotriazolyl carbonate (0.89 g of 70% mixture, 0.00210 moles)were added and the reaction mixture was stirred overnight at roomtemperature under an argon atmosphere. The solvent was evaporated todryness using a rotary evaporator. The crude product was dissolved inmethylene chloride and precipitated with isopropyl alcohol. The wetproduct was dried under reduced pressure. Yield 10.0 g.

NMR (d₆-DMSO): 1.10 ppm (t, CH₃ CH₂—, 6H), 1.51 ppm (m,—CH₂CH₂-(butyraldehyde), 4H), 1.67 ppm (m, —CH₂ CH₂COO—, 2H), 2.12 ppm(t, —CH₂COO—, 2H), 3.12 ppm (q, —CH₂ NH—, 4H), 3.51 ppm (s, PEGbackbone), 3.99 ppm (m, —CH₂—O(C═O)—, 4H), 4.46 ppm (t, 1H, acetal),4.62 ppm (m, PEG-O—CH₂—O(C═O)O—, 4H), 7.19 ppm (t, —(C═O)NH—, 2H),7.41-8.21 ppm (complex mult., benzotriazole protons, 4H), 7.80 ppm (t,—(C═O)NH—, 1H). Substitution: ˜100%.

AHPDP-PEG2(10 KDa)-Butyraldehyde

To a solution of BTC-PEG2(10 KDa)-butyraldehyde, diethyl acetal (10 g,0.0010 moles) in anhydrous methylene chloride (100 ml),3-amino-1-hydroxypropane-1,1-diphosphonic acid, ditetrabutylammoniumsalt (AHPDP-2Bu₄N) (1.7 g) and triethylamine (3.0 ml) were added and thereaction mixture was stirred overnight at room temperature under argonatmosphere. Next solvent was evaporated to dryness. The crude productwas dissolved in DI water (200 ml) and the solution was filtered throughAmberlite IR 120 (plus) column (100 ml). Next the pH of the solution wasadjusted to 2.5 with a 5% H₃PO₄. The solution was stirred 3 h then thepH was readjusted to 6.6 with 1M sodium hydroxide. Low molecular weightcompounds were removed from the solution by ultrafiltration. Next waterwas distilled off under reduced pressure giving 6.2 g of white solidproduct.

NMR (d₆-DMSO): 1.75 ppm (m, —CH₂ —CH₂—CHO, 2H and —CH₂ CH₂COO—, 2H),2.02 ppm (m, —CH₂— (AHPDP), 4H), 2.10 ppm (t, —CH₂COO—, 2H), 2.44 ppm(dt, —CH₂ —CHO, 2H), 3.12 ppm (q, 4H), 3.51 ppm (s, PEG backbone), 4.03ppm (m, —CH₂—O(C═O)—, 4H), 7.19 ppm (t, —(C═O)—NH—, 2H), 7.80 ppm (t,—(C═O)—NH—, 1H), 9.66 ppm (t, —CHO, 1H).

Example 3 I. Preparation of Trilysine Based BzO-PEG4_((20K))-Acid

Trilysine (1.0 g, 0.00666 equivalents) (Sigma-Aldrich Corporation, St.Louis, Mo.) was dissolved in 0.1M boric acid solution (200 ml) and thepH of the solution was adjusted to 8.5 with 0.1M NaOH. NextBenzyloxy-PEG_((5K))-benzotriazolyl carbonate (BzO-PEG_((5K))BTC) (40.0g, 0.00800 moles) (Nektar Therapeutics, Huntsville, Ala.) was added over45 min during stirring. During BzO-PEG_((5K))BTC addition the pH waskept 8.5-9.0 by periodical addition of 0.1M NaOH. Then the reactionmixture was stirred overnight at room temperature. Sodium chloride wasadded (10 g) and the pH of the mixture was adjusted to 2.0 with 10%solution of H₃PO₄. The crude product was extracted with CH₂Cl₂. Theextract was dried (MgSO₄) and the solvent was distilled off underreduced pressure.

The crude product was purified by ion exchange chromatography asdescribed in U.S. Pat. No. 5,932,462 giving 22.8 g of 100% pure product.

NMR (d₆-DMSO): 1.10-175 ppm (complex mult., —CH—(CH₂ )₃— (lysine), 18H),3.12 ppm (q, —CH₂ —NH(C═O)—, 6H), 3.51 ppm (s, PEG backbone), 3.92 ppm(m, —CH—COOH, 1H), 4.03 ppm (m, —CH₂O(C═O)NH—, 8H), 4.49 ppm (s, —CH₂—(benzyl), 8H), 7.14 ppm (t, —CH₂ NH(C═O)—, 3H), 7.32 ppm (m, —C₆H₅, 20Hand —CHNH(C═O)—, 1H).

2. Trilysine Based HO-PEG4_((20K))-Acid

Trilysine based BzO-PEG4_((20K)) acid (22.0 g, 0.00110 mole) (preparedas described above) was dissolved in ethyl alcohol (96%, 200 ml) andpalladium hydroxide on the active carbon (20 wt. % of Pd, water content50%; 1.5 g) was added and the reaction mixture was hydrogenated under 40psi of hydrogen overnight. Next the mixture was filtered and the solventwas distilled off under reduced pressure. The crude product wasdissolved in CH₂Cl₂ (300 ml). The solution was dried (MgSO₄) and thesolvent was distilled off under reduced pressure. The wet product wasdried under vacuum giving 19.5 g of white solid.

NMR (d₆-DMSO): 1.10-175 ppm (complex mult., —CH—(CH₂ )₃— (lysine), 18H),3.12 ppm (q, —CH₂ —NH(C═O)—, 6H), 3.51 ppm (s, PEG backbone), 3.92 ppm(m, —CH—COOH, 1H), 4.03 ppm (m, —CH₂O(C═O)NH—, 8H), 4.56 ppm (t, —OH,4H), 7.14 ppm (t, —CH₂ NH(C═O)—, 3H), 7.31 ppm (d, —CHNH(C═O)—, 1H).

3. Trilysine Based HO-PEG4_((20K))-Butyraldehyde, Diethyl Acetal

Trilysine based HO-PEG4_((20K))-Acid (20.0 g, 0.00100 mole) (prepared asdescribed above) was dissolved in anhydrous dichloromethane (200 ml) andtetra(ethylene glycol)-α-amino-ω-butyraldehyde, diethyl acetal (3.70 g,0.00110 moles) and 1-hydroxybenzotriazole (0.140 g, 0.00105 moles), andN,N-dicyclohexylcarbodiimide (2.30 g, 0.00111 mole) were added. Themixture was stirred overnight at room temperature under argonatmosphere. Next, part of the solvent was distilled off under reducedpressure and the product was precipitated with isopropyl alcohol at roomtemperature and dried under vacuum giving 19.5 g of white powder.

NMR (d₆-DMSO): 1.10 ppm (t, CH₃ CH₂—, 6H), 1.10-175 ppm (complex mult.,—CH—(CH₂ )₃— (lysine), 18H and —CH₂CH₂— (butyraldehyde), 4H), 3.12 ppm(q, —CH₂ —NH(C═O)—, 6H), 3.51 ppm (s, PEG backbone), 3.92 ppm (m,—CH—COO—, 1H), 4.03 ppm (m, —CH₂O(C═O)NH—, 8H), 4.46 ppm (t, —CH(acetal), 1H), 4.56 ppm (t, —OH, 4H), 7.14 ppm (t, —CH₂ NH(C═O)—, 3H),7.31 ppm (d, —CHNH(C═O)—, 1H). Substitution: ˜100%.

4. Preparation of Trilysine Based BTC-PEG4_((20K))-Butyraldehyde,Diethyl Acetal

To a solution of trilysine based HO-PEG4_((20K)) butyraldehyde, diethylacetal (19.5 g, 0.00390-OH equivalents) in anhydrous acetonitrile (200ml), pyridine (1.25 ml) and dibenzotriazolyl carbonate (3.30 g of 70%mixture, 0.007800 moles) were added and the reaction mixture was stirredovernight at room temperature under an argon atmosphere. The mixture wasfiltered and the solvent was evaporated to dryness using a rotaryevaporator. The crude product was dissolved in methylene chloride andprecipitated with isopropyl alcohol. The wet product was dried underreduced pressure. Yield 19.0 g.

NMR (d₆-DMSO): 1.10 ppm (t, CH₃ CH₂—, 6H), 1.10-1.75 ppm (complex mult,—CH—(CH₂ )₃— (lysine), 18H and —CH₂CH₂— (butyraldehyde), 4H), 3.12 ppm(q, —CH₂ —NH(C═O)—, 6H), 3.51 ppm (s, PEG backbone), 3.92 ppm (m,—CH—COO—, 1H), 4.03 ppm (m, —CH₂—O(C═O)—, 8H), 4.45 ppm (t, 1H, acetal),4.62 ppm (m, mPEG-O—CH₂—O(C═O)O—, 8H), 7.14 ppm (t, —CH₂ NH(C═O)—, 3H),7.31 ppm (d, —CHNH(C═O)—, 1H), 7.41-8.21 ppm (complex mult,benzotriazole protons, 16H). Substitution: ˜100%.

5. Preparation of Trilysine Based AHPDP-PEG2_((20K))-Butyraldehyde

To a solution of trilysine based BTC-PEG4_((20 K))-butyraldehyde,diethyl acetal (19 g, 0.0380—BTC equivalents) in anhydrous methylenechloride (300 ml), 3-amino-1-hydroxypropane-1,1-diphosphonic acid,ditetrabutylammonium salt (AHPDP-2Bu₄N) (6.4 g) and triethylamine (5.8ml) were added and the reaction mixture was stirred overnight at roomtemperature under argon atmosphere. Next solvent was evaporated todryness. The crude product was dissolved in DI water (400 ml) and thesolution was filtered through Amberlite IR 120 (plus) column (100 ml).Next the pH of the solution was adjusted to 2.5 with a 5% H₃PO₄. Thesolution was stirred 3 h then the pH was readjusted to 6.6 with 1Msodium hydroxide. Low molecular weight compounds were removed from thesolution by ultrafiltration. Next water was distilled off under reducedpressure giving 17.4 g of white solid product.

NMR (d₆-DMSO): 1.10-1.75 ppm (complex mult., —CH—(CH₂ )₃— (lysine),18H), 2.02 ppm (m, —CH₂— (AHPDP), 8H), 2.44 ppm (dt, —CH₂ CHO, 2H), 3.12ppm (q, —CH₂ NH(C═O)—, 4H), 3.51 ppm (s, PEG backbone), 3.92 ppm (m,—CH—COO—, 1H), 4.03 ppm (m, —CH₂—O(C═O)—, 8H), 7.14 ppm (t, 2H), 7.31ppm (d, 1H), 9.66 ppm (t, —CHO, 1H).

What is claimed is:
 1. A heterobifunctional, substantially linear,hydroxyapatite-targeting polymer having the structure:Z—(X¹)_(a)-L¹-(X²)^(b)-[POLY¹-(X³)_(c)-L²-(X⁴)_(d)]_(m)-POLY²-(X⁵)_(e)—Ywherein: each POLY¹ and POLY², which may be the same or different, is awater-soluble, non-peptidic polymer having a number average molecularweight of less than 8,000 Da; each X¹, X², X³, X⁴, and X⁵, which may bethe same or different, is a spacer moiety; L¹ is a linkage; each L² is ahydrolytically or enzymatically cleavable linkage selected from thegroup consisting of carbamate and amide; Z is a hydroxyapatite-targetingmoiety; Y is a functional group; each a, b, c, d, and e, which may bethe same or different, is either zero or one; and m is an integer in therange of 1-10.
 2. The polymer of claim 1, wherein each of X¹, X², X³,X⁴, and X⁵, when present, is selected from the group consisting of—C(O)O—, —OC(O)—, —CH₂—C(O)O—, —CH₂—OC(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—,—C(O)—, —C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—,—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—, —CH₂—C(O)—O—CH₂—,—CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, and—O—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —O—C(O)—NH—[CH₂]_(h)—(OCH₂CH₂)_(j)—,—NH—C(O)—O—[CH₂]_(h)—(OCH₂CH₂)_(j)—, bivalent cycloalkyl group, —O—,—S—, —N(R⁶)—, and combinations thereof, wherein R⁶ is H or an organicradical selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl andsubstituted aryl, (h) is zero to six, and (j) is zero to 20.